Spatial Patterns of Bird-Parasite Interactions Along an Urbanisation Gradient Carlos Andres Delgado-Velez University of Wollongong

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2015 Spatial patterns of bird-parasite interactions along an urbanisation gradient Carlos Andres Delgado-Velez University of Wollongong

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Spatial patterns of bird-parasite interactions along an

urbanisation gradient

A thesis submitted in fulfillment of the requirements for the

award of the degree

Doctor of Philosophy

From

University of Wollongong

Carlos Andres Delgado-Velez, BSc (Hons)

SCHOOL OF BIOLOGICAL SCIENCES

2015

Red-browed finch and Superb-fairy wren; passerine birds upon which this thesis is mostly based. They are affected differentially by parasites in urban areas.

CERTIFICATION

I, Carlos Andres Delgado-Velez, declare that this thesis, submitted in fulfillment of the requirements for the award of Doctor of Philosophy, in the Department of Biological

Sciences, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged below. The document has not been submitted for qualifications at any other academic institution.

Carlos Andres Delgado-Velez

25 March 2015

PUBLICATIONS

This thesis includes chapters that have been written as the following journal articles:

Chapter one: Delgado-V., C.A., French, K. 2012. Parasite-bird interactions in urban areas: Current evidence and emerging questions. Landscape and Urban Planning. 105,

5-14.

Chapter three: Delgado-V., C.A., French, K. In review. Urbanisation does not increase ectoparasite prevalence in Australian passerine birds. Urban Ecosystems.

Chapter four: Delgado-V., C.A., French, K. In press. Differential influence of urbanisation on Coccidian infection in two passerine birds. Parasitology Research [DOI:

10.1007/s00436-015-4414-2].

My supervisor, as co-author on my publications, has helped with data collection, analysis, interpretation and writing but this has not been above and beyond that which is expected of a PhD supervisor. Therefore I, as primary author in all mentioned papers, have been solely responsible for submitting each manuscript for publication to the relevant journals, and have been in charge of responding to reviewers’ comments.

For Chapter five, I also received contributions from Paulo Pulgarín, MSc. His contribution to this paper was focused on performing and advising on the technical aspects of the molecular screening.

STATEMENT OF CANDIDATE CONTRIBUTION

As the supervisor, I, Professor Kristine French, declare that the greater part of the work in each article listed is attributed to the candidate, Carlos Andres Delgado-Velez who led conceptual development, study design and was primarily responsible for data collection, data analysis, and data interpretation. Carlos has been solely responsible for submitting each manuscript for publication to the relevant journals, and he has been in charge of responding to reviewers’ comments.

______Professor Kristine French Supervisor 25 March 2015

______Carlos Andres Delgado-Velez Candidate 25 March 2015

CONFERENCES AND OTHER PUBLICATIONS

I have also presented data from this thesis at the following national and international conferences:

- Delgado-V., C.A., French, K., 2012. Are urban birds full of parasites? Ecological Society of Australia Conference. December 3-7. Melbourne, Australia.

- Delgado-V., C.A. 2012. Bird-parasite interactions along an urbanisation gradient. Postgraduate Student Conference, University of Wollongong. November 2. Kioloa, Australia.

- Delgado-V., C.A. 2011. Bird-parasite interactions in urban areas. Postgraduate Student Conference, University of Wollongong. November 3. Kangaroo Valley, Australia.

- Delgado-V., C.A., French, K., 2011. Spatial dynamics of bird-parasite interactions along an urban gradient. International Conference on Malaria and Related Haemosporidian Parasites of Wildlife. Malaria Research Coordinator Network. Agosto 5-7. National Conservation Training Center, Shepherdstown, West Virginia, USA.

In addition to the published manuscripts listed above, during the course of my studies as PhD student at University of Wollongong I have published two journal articles which were based on observations obtained during my studies in Australia:

- Delgado-V., C.A., Correa-H., J.C. Accepted. Sugar packet opening by the Noisy Miner: a novel foraging behavior. The Wilson Journal of Ornithology.

- Delgado-V., C.A., Correa-H., J.C. 2013. An unusual foraging tactic of the Willie Wagtail. The Wilson Journal of Ornithology. 125, 846-848.

TABLE OF CONTENTS

TITLE PAGE …………...... 1

CERTIFICATION ...... 3 PUBLICATIONS ...... 4 STATEMENT OF CANDIDATE CONTRIBUTION ...... 5 CONFERENCES AND OTHER PUBLICATIONS ...... 6 TABLE OF CONTENTS ...... 7 LIST OF TABLES ...... 10 LIST OF FIGURES ...... 12

ABSTRACT ...... 14 ACKNOWLEDGMENTS ...... 16 PROLOGUE ...... 18 RESEARCH AIMS ...... 21 OVERVIEW OF THIS THESIS ...... 22

CHAPTER ONE: PARASITE-BIRD INTERACTIONS IN URBAN AREAS: CURRENT EVIDENCE AND EMERGING QUESTIONS ...... 27 1.1. Introduction ...... 27 1.2. Literature Review ...... 30 1.2.1. Methods ...... 30 1.3. Results ...... 31 1.3.1. Urban areas, parasites and bird hosts ...... 32 1.3.2. Spatial patterns along an urbanisation gradient ...... 34 1.3.3. Parasite Effects on Host health ...... 48 1.4. Conclusions ...... 50

CHAPTER TWO: EVALUATION OF A COMMERCIAL INSECTICIDE POWDER FOR QUANTIFICATION OF BIRD ECTOPARASITES IN ECOLOGICAL STUDIES ...... 51 2.1. Introduction ...... 51 2.2. Methods ...... 54 2.3. Results ...... 56 2.4. Discussion ...... 58 2.5. Conclusions ...... 60

CHAPTER THREE: URBANISATION DOES NOT INCREASE ECTOPARASITE PREVALENCE IN AUSTRALIAN PASSERINE BIRDS ...... 61 3.1. Introduction ...... 61 3.2. Methods ...... 63 3.3. Results ...... 67 3.4. Discussion ...... 70 3.5. Conclusions ...... 72

CHAPTER FOUR: DIFFERENTIAL INFLUENCE OF URBANISATION ON COCCIDIAN INFECTION IN TWO PASSERINE BIRDS ...... 73 4.1. Introduction ...... 73 4.2. Methods ...... 76 4.3. Results ...... 77 4.4. Discussion ...... 79 4.5. Conclusions ...... 82

CHAPTER FIVE: PREVALENCE AND LINEAGE DIVERSITY OF AVIAN HAEMOSPORIDIANS FROM URBAN AND NATURAL AREAS IN EASTERN AUSTRALIA ...... 83 5.1. Introduction ...... 83 5.2. Methods ...... 86 5.2.1. Field sites ...... 86 5.2.2. Bird capture and handling ...... 87 5.2.3. Analysis and measurements of blood parasite infection by microscopy ...... 87 5.2.4. Molecular detection ...... 88 5.2.5. Literature review ...... 89 5.3. Results ...... 90 5.3.1. Overall urban Haemosporidian prevalence by microscopy screening ...... 90 5.3.2. Comparison of prevalence and lineage diversity between urban and natural areas using sequencing ...... 90 5.3.3. Literature review ...... 91 5.4. Discussion ...... 103 5.4.1. Are Australian Haemosporidians distinct? ...... 103 5.4.2. Microscopy versus Molecular techniques for sampling Haemosporidians ...... 105 5.4.3. Are prevalence levels similar to other parts of the world? ...... 105 5.4.4. Do urban areas have greater prevalence of Haemosporidians? ...... 106

5.5. Conclusions ...... 108

CHAPTER SIX: SPATIAL CHANGES IN HEALTH PARAMETERS AND BODY CONDITION OF URBAN SENSITIVE BIRDS ALONG AN URBAN GRADIENT ...... 110 6.1. Introduction ...... 110 6.2. Methods ...... 112 6.2.1. Field sites ...... 112 6.2.2. Sampling ...... 114 6.2.3. Body condition ...... 114 6.2.4. Haematological condition ...... 114 6.2.4.1. Haematocrit levels and Haemoglobin concentrations ...... 115 6.2.4.2. Heterophil:Lymphocyte ratio and total leucocyte count ...... 115 6.2.5. Statistical analysis ...... 116 6.3. Results ...... 117 6.3.1. Body condition ...... 117 6.3.1.1. Body mass ...... 117 6.3.1.2. Head and bill length ...... 117 6.3.2. Haematological condition ...... 117 6.3.2.1. Haemoglobin concentrations ...... 117 6.3.2.2. Haematocrit ...... 118 6.3.2.3. H/L ratio ...... 118 6.3.2.4. Total Leukocyte Count ...... 119 6.4. Discussion ...... 125 6.5. Conclusions ...... 129

CHAPTER SEVEN: CONCLUSION, GENERAL DISCUSSION AND FUTURE DIRECTIONS ...... 130

REFERENCES ...... 133

LIST OF TABLES

CHAPTER ONE

Table 1.1. Summary of the descriptive research about the diversity and occurrence of parasites on urban birds………………………………………..…………………………………….……36

Table 1.2. Summary of studies investigating spatial patterns of parasite-bird interactions……………………………………………………………………….….………….39

Table 1.3. Summary of studies on parasite effects on birds in urban centres……….….…...…41

CHAPTER TWO

Table 2.1. Mean ± SD, range and total numbers of feather mites, lice, ticks and louse flies recovered from 72 passerines using visual examination and Pestene dust-ruffling methods……………………………………………………………….…….……………..….....57

Table 2.2. Differences in prevalence and mean intensity of mites, lice, flies and ticks from a sample of 72 birds using visual examination and the Pestene dust-ruffling………….….....…..58

CHAPTER FIVE

Table 5.1. Haemosporidians found in blood smears from birds from Illawarra urban areas. The list is ordered alphabetically by bird familiy. N (number of individuals sampled), I (number of individuals infected) (Prevalence in %), L (Leucocytozoon), H (Haemoproteus), P

(Plasmodium)……………………………………………………………………..………..…....93

Table 5.2. Prevalence (% of birds with infection) of Haemosporidians with no specific parasite discrimination (TH), Plasmodium/Haemoproteus (P/H), and Leucocytozoon (L) infections for

Superb-fairy-wrens and Red-browed finches in urban and natural sampling sites…...………..94

Table 5.3. MalAvi accession numbers of lineages identified in this study…………………...... 94

Table 5.4. Area and number of individuals of Superb-fairy wren (SFW) and Red-browed finch

(RBF) infected by each mitochondrial lineage of Haemoproteus and Leucocytozoon parasite...... 94

Table 5.5. Haemosporidians prevalence data obtained for natural sites located in the Southern

Hemisphere recovered from the literature review. Abbreviations include: Type of

Haemosporidian (P: Plasmodium, H: Haemoproteus, L: Leucocytozoon) and P (Prevalence in

%)………………………………………………………………………………………...….….95

Table 5.6. Haemosporidian prevalence data of urban areas around the world recovered from selected references. Abbreviations include: Type of Haemosporidian (P: Plasmodium, H:

Haemoproteus, L: Leucocytozoon) and P (Prevalence in %)...... 100

LIST OF FIGURES

CHAPTER ONE

Figure 1.1. Number of publications and main type of research carried out in four continents on parasite-bird interactions in urban areas. No urban-parasite research was published for

Africa…………………………………………………………………………………..………..33

CHAPTER THREE

Figure 3.1. Map displaying the ectoparasite sampling sites locations………………………….68

Figure 3.2 a. Lice, b. Ticks and c. Louse flies prevalences for Red-browed finch (■), and

Superb-fairy wren (♦) along an urban gradient. Best fit lines for RBF (···) and SFW (––) are depicted when the degree of urbanisation exhibited significant effects………………………...69

CHAPTER FOUR

Figure 4.1. Coccidian prevalences for a. Red-browed finch and b. Superb-fairy wren in breeding (♦), and non-breeding seasons (■), along an urban gradient. Best fit lines for RBF are depicted. Degree of urbanisation exhibited no significant effects in SFW……………………..78

CHAPTER SIX

Figure 6.1. Variation of average body mass of a. Red-browed finch and b. Superb-fairy wren along sites with different degrees of urbanisation development………………………………119

Figure 6.2. Variation of average head and bill length of a. Red-browed finch and b. Superb- fairy wren along sites with different degrees of urbanisation development…………………..120

Figure 6.3. Variation of average Haemoglobin concentration of a. Red-browed finch and b.

Superb-fairy wren along sites with different degrees of urbanisation development…………..121

Figure 6.4. Average Haematocrit level of Red-browed finch in a. Breeding and b. Non Breeding season, and c. Superb-fairy wren along sites with different degrees of urbanisation development…………………………………………………………………………………....122

Figure 6.5. H/L ratio of a. Red-browed finch and b. Superb-fairy wrens females and c. males in

Breeding season along sites with different degrees of urbanisation development…………….123

Figure 6.6. Variation of Total Leukocyte Count of a. Red-browed finch and b. Superb-fairy wren along sites with different degrees of urbanisation development………………………...124

ABSTRACT

Urbanisation is a fast growing phenomenon worldwide which has direct and indirect impacts on biodiversity. Urbanisation affects particular species but also biotic relationships and interactions. Changes in host-parasite interactions have the potential to be one of the important consequences of urbanisation, influencing the species able to exploit, establish and succeed in urban landscapes. However, how urbanisation influences the prevalence and impact of parasites and the susceptibility of different birds has not been studied in detail, especially in areas such as Australia and many Oceanic islands. The risk of parasitism is predicted to increase in cities due to changes in climatic conditions, habitat loss, habitat conversion, increased temperature, fragmentation and a greater presence of human-modified landscapes.

Integrating ecological and physiological approaches, this thesis analyses the distribution and diversity of three groups of bird parasites along an urbanisation gradient in New

South Wales, Australia, and investigates how parasites and haematological and body condition may influence the structure of urban-bird communities. Given the paucity of information available in Australia, this thesis provides an inventory of a portion of

Australia parasite fauna and tested whether parasite loads could influence the ability of some birds to inhabit cities. I particularly focused on two species of birds which are not particularly common in urban areas.

Contrary to expectations, my research does not support a positive association of urbanisation and bird parasites, nor does it identify detrimental effects on the immune system and body condition of the host. Instead, results suggest that urbanisation does

14 not necessarily result in high parasitism and lower immune system in urban-sensitive birds. This thesis found that some parasites such as louse flies, ticks and haemosporidians are less prevalent (or even disappear) while others might be either more prevalent (e.g. Coccidian parasites) or kept unchanged in urban areas (e.g. lice).

My study was unable to detect a general pattern of loss of body condition associated with urban development for the species sampled suggesting that urbanisation effects are complex and might be site-, parasite- and host-specific related.

This thesis identified four new lineages of haemosporidians, indicating great potential for Australian bird parasites to be fundamentally unique. The poor knowledge available on the relationship among parasites and wildlife still requires further consideration across more species in order to determine how parasites are affected by urbanisation and whether they play a role in the establishment and success of birds in cities. At present parasite load does not appear to be a cause of the decline in urban birds.

ACKNOWLEDGMENTS

First of all, I would like to thank my supervisor Kris French for accepting me as her student and encouranging me to complete the crazy experience of conducting a PhD project overseas. As an international student new in such an immense and distant country, Kris is the kind of tutor everyone would always wish to have, not just for her insight and support to my research whenever needed, but also for her mood, generosity and unconditional kindness, aspects that were crucial to stay in and complete this process.

There were many people who helped with particular aspects of my research and time in

Australia. Personally, I received generous and disinterested help from Australians: I thank Danina Scrivenor and her family for helping us to settle well and safe. Jocelyn and Ted Booth lent us their bicycles which allowed Juanis and I to transport around

Wollongong with independence. Kel Magrath and, Jacqui and David Price for the warm hospitality and company they offered us. The members of the French' Lab for friendship and lab and field support. Moreover, I would like to thank Daniel Zuleta for providing advice on statistical analysis and Paulo Pulgarín for assisting with molecular screening.

This study could not have been completed without the enduring support of my family and friends in -or coming from- my natal country: Juanis offered me endless patience, love, help and company; “mi mamá” who took care of Fênix with such love during my time in Australia, ¡loquito perdón por abandonarte tanto tiempo!; thanks to Chucho for his crucial moral support while finishing and submitting the thesis; and all the “viejitos” for being always close to me.

Funding was received from The Joyce Vickery Scientific Research Fund and The

Linnean Society of NSW. All urban field trips were carried out on a Yuba Mundo bicycle which was partly donated by CargoCycles (Australia) and Yuba Bicycles (USA) for conducting urban ecology studies. Finally, I received scholarships from University of Wollongong. Without them, I would have never visited Australia to study and run with kangaroos.

PROLOGUE

Urban areas are increasing in both extent and number (Evans et al. 2009) which is causing important impacts on biodiversity through a range of processes. However, while the effects of urbanisation on biodiversity structure and composition have been quite well studied, interactions and relationships amongst species are virtually unknown

(Schochat et al. 2006).

Urbanisation causes significant changes to species composition, species interactions and ecological and evolutionary processes (Gaston 2010). Changes in host-parasite interactions have the potential to be one of the important consequences of urbanisation, and influence, perhaps, the species ability to exploit urban landscapes (Martin and

Boruta 2014; Riley et al. 2014). However, how urbanisation influences the prevalence and impact of parasites and the susceptibility of different birds to them has not been studied in detail.

Some bird species survive and prosper in urbanised areas (“Urban Tolerant Birds”), but others are directly or indirectly affected by a wide range of anthropogenic activities and infrastructure (“Urban Sensitive Birds”) (Parsons et al. 2006). An increase in some kinds of food and water, reduced presence of predators, fewer or limited competitive interactions, and a changed microclimate influence the inclusion or exclusion of birds in cities (Fokidis et al. 2008), however they do not explain (or characterise) fully the presence, establishment and success of some native species that thrive in urban environments around the world. Therefore some other significant factors that are more cryptic, and unstudied may be important in elucidating the causes

18 of presence (and absence) of species in urban areas.

Parasites are a dominant and diverse group which is fundamental in ecological and evolutionary aspects (Atkinson et al. 2008). For example, certain parasitic life strategies may allow the maintenance of ecological processes and will be critical to the conservation of fully functioning ecosystems in natural environments (Nichols and

Gómez 2011). However, chronic alterations in parasite prevalence might cause significant changes in vertebrate hosts and change ecological processes.

Birds are host to a variety of parasites which exhibit a diverse array of form and life history, and where obligate and non-obligate taxa are recognised as vectors of pathogens and diseases (Pershikova et al. 2010). Avian blood parasites and ectoparastites, but also others protozoa, bacteria, viruses, cestodes and nematodes may play important roles such as increasing mortality (Ludwing et al. 2010); reducing fecundity (Heins and Baker 2014); altering nutritional status (Aronstein et al. 2012); reducing growth (Mouritsen and Poulin 2002); enhancing susceptibility to predation

(Laferty and Morris 1996); altering mate choice (Minchella and Scott 1991); and affecting body condition (Faim et al. 2008). Therefore, parasitism could contribute greatly to the structure, diversity, conservation, function, and health of urban biodiversity (McCallum and Dobson 1995).

Parasites and pathogens may cause several detrimental effects in native species globally

(Daszak et al. 2000; Cumming and Van Vuuren 2006). Since the risk of parasitism and wildlife diseases caused by ecto- and endoparasites are likely to increase due to human activity (Bradley and Altizer 2006), parasites may be an important factor affecting the

19 presence of wildlife species in cities. However, in contrast to the growing interest in studying factors that can influence the risk of parasitism inherent to the host, such as sex, age, reproductive status and behaviour, the habitat-specific factors that promote parasitism have been mostly ignored (Kleindorfer et al. 2006). As a result, we have very little understanding of whether parasite infestations in disturbed habitats such as cities, influence the host species that can exist in these habitats.

The prevalence and impact of wildlife diseases and parasite loads in urban areas differ amongst studies (Bradley and Altizer 2006). Some authors have found that urban centres have lower rates of parasite infection than rural or natural areas due to, for example, an increase in food availability which probably helps urban birds to create resistance through increased immunity (Gregoire et al. 2002). In contrast, others suggest a positive relationship of abundance and/or prevalence of parasites in urban areas (e.g.

Cumming and Van Vuuren 2006, Morand and Poulin 1998).

Avian urban parasite studies have been carried out preferentially in Europe and North

America (Delgado-V. and French 2012). Moreover, such studies have been focused on a couple of species which are abundant in cities such as European blackbirds, House sparrows and crows. As a result, although there are globally few studies to help elucidate the role of parasites in urban areas, especially in Australia, most of them are focused on Urban Tolerant Birds from temperate locations but just few have been carried out on Urban Sensitive Birds (Fokidis et al. 2008).

At present therefore, studies have not been undertaken on the groups of birds for which parasitism may be the cause of their decline in cities. Additionally, since different bird

20 species are indeed differentially susceptible to parasite infestation (e.g. P. reli c t u m causes diseases of different severity in different passerine birds, Palinauskas et al.

2008), the consequences of parasite host dynamics due to anthropogenic environments should be explored. This means that a comparative analysis of parasite load should also be linked with measures of health in birds since species which are common in cities may be advantaged by increased immunity whereas species which are now rare in urban environments may be more susceptible. Thus different species of birds may have different susceptibility to parasite load thus influencing their ability to occupy urban centres.

This PhD project investigates the dynamics of bird-parasite interactions in the Illawarra and Sydney regions to determine the relationship between infestation loads, health and body condition in order to determine how parasites and immune status can change and potentially affect bird communities along a gradient of urbanisation.

RESEARCH AIMS

This thesis seeks to describe parasite load and the host body condition and susceptibility along urban gradients, to investigate their importance in contributing to the decline of some passerine birds in Australia (Ford et al. 2001). I hypothesized that Urban Sensitive

Species may be less exposed to parasites in natural habitats where they are in better condition and have better immune systems and where parasites are less prevalent. While not all species occur in every habitat, comparisons in parasite load and health of birds in habitats where they are rare will identify if parasite load increases or decreases differentially with urbanisation.

Specifically, I will

1. Describe the diversity and direction of the bird-parasite relationships identifying prevalence of bird parasites in habitats with different degree of urbanisation development;

2. Examine the immune system and body condition of two Urban Sensitive Species; and

3. Determine if two Urban Sensitive Species are more prone to parasites in cities.

OVERVIEW OF THIS THESIS

One of the chapters (chapter one) has already been published (Delgado-V. and French

2012). Additonally, chapter four is in press (Delgado-V. and French in press). The rest of data chapters are written in the format of separate scientific papers for submission to peer-reviewed journals (for example, chapter three was submitted to Urban

Ecosystems). Therefore there is some inevitable repetition of content between chapters.

This thesis is the original work of the author, unless stated (see Publications). I provide a brief overview of each chapter

Chapter one: I compile information about bird-parasite interactions publications in urban areas around the world. This review of the literature identified a poor knowledge of this interaction. While studies identifying parasites were more common than those comparing parasite abundance or host health, studies were limited geographically and to

22 a few bird species. I found that urbanisation could have a positive or negative effect on the diversity and prevalence of bird parasites. Very few studies have linked parasite loads to bird health. I identify some hypotheses that need further consideration across broader regions and species to determine if parasites play a role in the establishment and success of birds in cities and how their health and body conditions are affected

(Delgado-V. and French 2012).

Chapter two: I performed a brief evaluation of the dust-ruffling method using Pestene, an insecticide powder used in veterinary practices but never tested in ecological field studies. Comparison of the visual examination and dust-ruffling methods for quantification of ectoparasite infestation carried out on 13 species captured suggested that the method using Pestene is significantly more sensitive in recovering a greater number of mites and lice. Also it allowed the identification of a greater proportion of the population that is infested by mites, lice and ticks and discerned a higher number of parasites per bird. Pestene is recommended as a valuable tool to measure prevalence and intensity of lice, mites and ticks infestation and provides a sound reason for improving estimates of ectoparasite prevalence and intensity in ecological studies.

Chapter three: Urbanisation has the potential to alter the risk of parasitism on wildlife as it is associated with habitat modification, fragmentation and decreased biodiversity.

As birds are commonly found in urban areas, they provide a valuable opportunity to study parasitism in urban environments. Native birds were examined along an urbanisation gradient for three groups of ectoparasites in order to detect if variation of prevalence might be directly related to urbanisation development. Prevalence of parasites declined or fluctuated with urbanisation, rather than increased. Lice were

23 equally prevalent regardless of the level of urban development and higher prevalence of ticks and louse flies seemed to be negatively correlated with urban intensity. Although parasite values are usually variable amongst hosts, parasite parameters exhibited similar trends within the hosts sampled which suggests that urbanisation does not necessary result in high ectoparasitism. Instead, urbanisation effects on bird parasites might be site-, parasite- and host-specific. Studies on other urban areas, seasons, parasites and a wider diversity of hosts are needed in order to understand the ecological distribution of parasitism in an urbanised world.

Chapter four: Urbanisation has the potential to increase the risk of parasitism on wildlife. Although some ectoparasite groups appear unaffected, different responses are hypothesized for parasites with simpler life histories such as gastrointestinal parasites.

Two native passerine birds affected by urbanisation were examined for Coccidian parasites along an urbanisation gradient in New South Wales, Australia, in order to detect if prevalence might be directly related to the degree of urbanisation. Prevalence of Isospora increased in more urbanised areas on Red browed Finch but this did not significantly change with the degree of urbanisation in Superb-fairy-wren. Diet, behaviour, and habits, are suspected to be the most influential factors on the variation seen between both species with the granivorous and gregarious species being significantly infected. Since the dynamics of urban wildlife-pathogen interactions is largely unexplored, more studies are needed to corroborate if this pattern of Isospora infections can be extended to other passerine birds in cities from Australia and overseas.

Chapter five: Australia is one of the poorest sampled regions for parasites. Such absence of knowledge prevents an understanding of the ecology and diversity of its

Haemosporidian fauna (globally as well as regionally), and the effects of different forms due to disturbance (such as the ones due to urbanisation) on the complex host-parasite relationship. In order to better understand the prevalence and diversity of

Haemosporidian parasites in eastern Australia, natural and urban sites were sampled in the Illawarra region, NSW. Of the 270 passerine birds screened using light microscopy in urban areas, a total of 0.7% were found infected, although the degree of infection varied among species. Additionally, molecular screening of 147 individuals of Superb- fairy wrens and Red-browed finches suggested that there are at least four undescribed lineages of Leucocytozoon and Haemoproteus, two respectively, and that their prevalence is higher or exclusive in natural areas. The few molecular studies of

Haemosporidians in Australia do not allow to conduct a deeper analysis, however I suspect that vectors might be implicated as response since, for instance, the absence of

Haemoproteus in urban areas might be due to the decrease of louse flies, one of its main vectors, in highly urbanised areas, as it was recorded in a concomitant study of ectoparasites which included the same sampling areas. Prevalence and diversity data here presented for first time in Eastern Australia have important insights into the epidemiology, diversity and transmission dynamics of Haemosporidian parasites in the country, even in highly disturbed areas.

Chapter six: Urban development is associated with activities and land uses changes that can cause detrimental changes in body conditions and immunology stages making wildlife more susceptible and vulnerable to different parasite pressures and diseases found in urban areas. In order to try to detect if susceptibility might be considered a factor affecting some bird species living in cities, I tested whether the degree of urbanisation affects the health and body conditions of the Red-browed finch and

Superb-fairy wren, two passerine birds considered affected by urbanisation in Australia.

I monitored six blood and body condition parameters along an urban gradient and found significant differences in Haemoglobin concentration, head and bill length, H/L ratio and Total Leukocyte Count among sites. For example, Haemoglobin concentration in

RBF is significant higher in natural areas while finches captured in industrial areas exhibited the highest value of H/L ratio. Contrary, the differences I detected for males and females of SFW were more subtle and restricted to H/L ratio in breeding seasons.

Results suggest that while wrens might have a greater capacity to cope well with parasite infections, pathogens and diseases in urban areas, the immune system of urban finches might not be in optimal condition to respond successfully to urban pressures in cities.

Chapter seven: Here I draw together the results from previous chapters together and discuss their implications and conclude the thesis.

CHAPTER ONE: PARASITE-BIRD INTERACTIONS IN URBAN AREAS:

CURRENT EVIDENCE AND EMERGING QUESTIONS

A modified version of this chapter is published in Landscape and Urban Planning.

Delgado-V., C.A., French, K., 2012. Parasite-bird interactions in urban areas: Current evidence and emerging questions. Landscape and Urban Planning. 105, 5-14.

1.1. Introduction

Urbanisation is a fast-growing phenomenon which is recognised as the major cause of biotic homogenisation, a process characterised by a loss of diversity and a concomitant progressive increase in similarity where the abundance of some species can increase while others generally decline (McKinney 2006). Urban growth has direct and indirect impacts on biodiversity with an increasing number of studies detecting detrimental effects on diversity of plants, arthropods, amphibians, birds, and mammals in temperate

(Chace and Walsh 2006; McKinney 2008) and tropical countries (Lim and Sodhi 2004;

Biamonte et al. 2011; Ortega-Álvarez and MacGregor-Fors 2011). For birds, highly urbanised places generally exhibit a high abundance of some groups, but an overall poor diversity, compared with adjacent natural areas (Blair 1996; Biamonte et al. 2011; Faeth et al. 2011), although some studies have identified a higher abundance in sites of intermediate development (Tratalos et al. 2007).

Urban ecology studies have been numerous in recent years (Schochat et al. 2006) but little emphasis has been given to the effects of urbanisation on ecological and 27 evolutionary processes (Alvey 2006; Chace and Walsh 2006; Christie and Hochuli

2009; Sumoski et al. 2009). However, urbanisation influences ecological interactions

(Faeth et al. 2005, 2011; Parson et al. 2006) such as pollination (Neil and Wu 2006) and seed dispersal (Cheptou et al. 2008), as well as competition and predation among vertebrates (Faeth et al. 2011). It might also significantly affect the interaction between parasites and their hosts causing changes in host composition.

Although parasites contribute to biodiversity (Hudson et al. 2006), they are causative factors of distress and vectors of disease for wild communities (Atkinson et al. 2008).

Previous reviews have summarised parasite-bird research for natural areas (e.g. Boyd,

1951; Mackerras and Mackerras 1960; Forrester and Spalding 2003), but not identified how such interactions are influenced by anthropogenic land change. Changes in parasite loads in response to urbanisation can influence the prevalence (proportion of infected hosts) and impact of wildlife diseases in urban areas (Bradley and Altizer 2006).

Changes might be influenced by a range of features in the urban environment including the increased presence of exotic species (Boal et al. 1998), poorer quality of habitat

(Patz et al. 2004), potential transference from domestic and exotic animals (Lehrer et al.

2010) and climate change, for example through warmer conditions (Cumming and Van

Vuuren 2006) associated with urban heat islands (Alekseev 2006). Thus, identifying parasites which are potentially affected by urbanisation is important in understanding urban biodiversity.

Changes in host-parasite interactions have the potential to influence which species are capable of inhabiting urban areas and the susceptibility of different species to parasites is likely to influence survival and presence in cities. Despite this, our understanding of

28 these interactions as a process structuring urban wildlife communities appears limited.

In this review, I gather the current knowledge about the relationship between ecto-, blood and gastro-intestinal parasites and one particular host taxon; birds. I base my review on birds as hosts since they are the vertebrate group best studied in cities around the world, are ubiquitous, have been used to explore effects and responses to urban development, and exhibit a complex community system in cities. They are used as bio- indicators of environmental quality in cities (Chace and Walsh 2006; Ortega-Álvarez and MacGregor-Fors 2011) and as sentinels for diseases and pathogens (Ludwig et al.

2010).

2007). Avian blood parasites and ectoparasites, but also others protozoa, bacteria, viruses, cestodes and nematodes may play important roles in sexual selection and reproductive success in birds (McCallum and Dobson 1995; Atkinson et al. 2008).

Therefore, parasitism could contribute greatly to the structure, diversity, conservation, function, and health of urban biodiversity.

In this review I seek to answer three specific questions about the relationship between parasites and urban birds:

1. What parasite fauna has been recorded on urban birds?

2. Are parasites, in terms of their intensity, diversity and prevalence, a relevant factor

29 influencing the establishment and success of birds in cities?

3. How is health and body condition of urban birds related to their parasite infestation?

Throughout the review I identify future areas of research and emerging questions based on responses obtained for these specific questions in order to better understand how urbanisation as a form of extreme disturbance, biodiversity homogenisation and deforestation (although the level of urbanisation and associated characteristics of a particular region or country might differ in intensity and extension, Gaston 2010), affects bird-parasite interactions.

Since parasites are also associated with transmission of infectious pathogens to humans and other fauna (Haag-Wackernagle 2005; Keesing et al. 2010; Reye et al. 2010; Sehgal

2010), my review could also have implications for wildlife management and public health in urban areas where, for example, exotic species, such as rock doves, come into closer contact with humans (Bart et al. 2008) or where wildlife may be a source of disease for domestic animals (Lehrer et al. 2010). In this sense, comparisons of bird/host interactions in natural and urbanised areas can detect human risks (Daszak et al. 2000) as well as detecting changes in this fundamental ecological interaction.

1.2. Literature Review

1.2.1. Methods

An intensive literature search was performed in the databases PubMed, Web of Science,

Biological Abstracts, Summon and Google Scholar from February to October 2011. A multi-keywords field search (in English and correspondent Spanish translations) included the following terms: “parasite*”, “pathogen*”, “disease*”, “bird*” and

“urban*, always using AND as a connector, and * as a proper truncation sign on each database. After this general search, a more specific one was performed adding speciﬁc parasites known to be associated with birds in wild areas such as “haemoparasite*” or

“ectoparasite*”. I also screened articles referenced by the initial identified publications in order to expand our literature. Research carried out on pets, poultry, seabirds (which have a recent review of their parasite fauna, Quillfeldt et al. 2011), or any captive manipulative study or laboratory trial in zoos or animal facilities were excluded from analysis although they were carried out within urban centres. I did not include brood parasitism by vertebrates in our review. Although this is a bird-related parasitic interaction which is widely recognised to be altered by anthropogenic factors, including urbanisation (Schmidt and Whelan, 1999), I omitted this particular mode of vertebrate- vertebrate interaction.

1.3. Results

My search gathered 73 papers dealing with issues related with one or more search topics in urban areas.

According to my questions, I categorised papers into three groups: 1. Descriptive

Studies (Table 1.1), which include research that recognises and cites the occurrence of bird parasites in urban areas; 2. Spatial patterns (Table 1.2), which includes research that compares parasite parameters such as prevalence and diversity of parasites in urban

31 with adjacent rural or natural areas; and 3. Parasite Effects (Table 1.3), which summarises multi-approach studies that seek to determine correlations between parasite load and the condition or health status of their urban bird host.

Descriptive Studies were the most common (Figure 1.1). There were no Spatial Pattern studies from Oceania, no Parasite Effect studies from either Asia or Oceania and no urban bird-parasite research published for Africa (Figure 1.1).

1.3.1. Urban areas, parasites and bird hosts

Information was recovered for 24 countries, and about 60 cities (Table 1.1). The parasite fauna is better known for American and European urban birds (Table 1.1), however, data are limited and restricted to few bird hosts from cities located in a limited geographic region. For example, Brazilian (9 studies) and US cities (17 studies) are centres for research within the Americas. The tropics were less well known (12 studies) than the temperate regions (42 studies) (Table 1.1).

25 Parasite effects Spatial Patterns 20 Descriptive

Number ofpublications 10

0 America Europe Asia Oceania

Figure 1.1 Number of publications and main type of research carried out in four continents on parasite-bird interactions in urban areas. No urban-parasite research was published for Africa.

Efforts to identify and develop inventories for haemoparasites and ectoparasites have received more attention than other types of parasites, but there is a growing interest in avian bacteria and viruses (Table 1.1) due to the potential of human transmissions

(Bradley et al. 2008). Despite this, knowledge is still rudimentary for all groups. For example, Ixodes ticks have been studied more than other arthropod groups (Bardach

1981; Gregoire et al. 2002; Arzúa et al. 2003) probably because they can be detected easily by visual examination (Gregoire et al. 2002), but new descriptions and biogeographic extensions of bird parasites still take place (e.g. Barros-Battesi et al.

2003), suggesting the field is in its infancy.

Inadequate baseline information on parasites in urban areas will hamper our understanding of patterns of host infestation and the role of parasites in cities. Few bird species have been studied and only in a few urban areas (Arzúa and Barros-Battesti

1999). Most studies have focused on well-adapted or introduced species (categorised as urban adapters and exploiters, Kark et al. 2007), such as rock doves (Marques et al.

2007) probably because they are ubiquitous, abundant and generally associated with zoonotic diseases (Zarrin et al. 2010). Very little data have been obtained for whole communities or for species that are more sensitive to urbanisation. The study by Arzúa and Barros-Battesi (1999) is one of the exceptions. They studied parasitism of ticks on birds in a Brazilian city providing new host records. However, for parasites with multiple hosts, few studies have identified secondary hosts and parasite prevalence in urban areas (Jansen et al. 2009).

1.3.2. Spatial patterns along an urbanisation gradient

Comparative data on the prevalence and intensity of infection by ectoparasites, blood parasites and other parasites in urban and natural populations of birds are mostly available for Europe and America (Figure 1.1). These provide insights about patterns of parasitism in cities, and whether they are possible factors influencing urban colonisation by birds. Some studies on other vertebrates in urban centres have been undertaken

(Skoracki et al. 2003).

The studies identify two different trends of parasite abundance along gradients from natural and/or rural to more urban developed areas. Some studies detect lower

34 parasitism in urbanised areas (compared with adjacent rural and natural areas) while others found a greater prevalence and intensity of infection by bird parasites in the more urbanised places (Table 1.2). Results apparently vary for city, host studied and type of parasite considered. For example, while Geue and Partecke (2008) found lower blood parasite prevalence for urban blackbirds in Munich, Germany, Boal et al. (1998) found a higher prevalence of other protozoa (Trichomonas) in urban hawks in Tucson, USA.

Differences in parasite prevalence might be explained by the type of parasite studied and their way of transmission. While malaria is a vector-borne disease, Trichomoniasis is transmitted by direct (for example through predation on infected birds which is case for raptor birds) or indirect (through a common use of water resources or feeding stations) contact (Atkinson et al. 2008). Therefore, a general pattern about how parasites are affected by urbanisation was not detectable in my review and identifies a significant research gap.

A further explanation for differences in observed urban patterns is the possibility that some studies may not detect microscopic parasites using blood smears (Braga et al.

2011). For example, while Evans et al. (2009) found a reduced prevalence of blood parasites in 8 out of 11 European cities studied using microscopy as the method of detection, Belo et al. (2011) found higher prevalence in Brazil using microscopy and molecular methods. While microscopy has certainly been used for urban studies and is considered a powerful tool for the study of haemosporidians (Braga et al. 2011), future research should include molecular techniques, to get more comprehensive prevalence and diversity measures for blood parasite in urban areas.

Table 1.1. Summary of the descriptive research about the diversity and occurrence of parasites on urban birds.

Country City Parasite Type Host Reference Argentina Córdoba Virus Furnarius rufus Tauro et al. 2009 Argentina San Juan Ectoparasites Columba livia Vallve et al. 1995 Argentina Corrientes Ectoparasites Columba livia Bar et al. 1993 Australia Gold Coast Bacteria and virus Threskiornis molucca Epstein et al. 2007 Australia Cairns, Brisbane, Newcastle and Blood parasites Various spp Jansen et al. 2009 Sydney Austria Vienna Ectoparasites Columba livia Bardach 1981 Brazil Lages Ectoparasites, Blood Columba livia Marques et al. 2007 parasites, and Gastro- intestinal parasites Brazil Saõ Paulo Fungus Columba livia Montenegro and Paula 2000 Brazil Curitiba Ectoparasites Various spp Arzúa and Barros-Battesti 1999 Brazil Serrinha, Mata de São João and Toxoplasma Passer domesticus Gondim et al. 2010 Coceição Brazil Jaboticabal Bacteria, Virus and Columba livia de Sousa et al. 2010 Toxoplasma Brazil Saõ Paulo, Bauru, Soracata, and Bacteria and Toxoplasma Columba livia de Lima et al. 2011 Botacatu. Brazil Pelotas Gastro-intestinal Columbina picui Coimbra et al. 2009 Brazil Porto Alegre Gastro-intestinal Passer domesticus Calegaro and Amato 2010 Brazil Brasília, Jataí and Uberlândia Blood parasites Passer domesticus Lima et al. 2010

Canada Quebec Virus Corvus brachyrhynchos Ludwig et al. 2010 Chile Santiago Bacteria, Virus, Columba livia Toro et al. 1999 Ectoparasites, Blood parasites, and Gastro- intestinal parasites. Croatia Zagreb Bacteria Columba livia Prukner-Radovcic et al. 2005 Croatia Zagreb Bacteria and Fungi Corvus frigilegus Gomercic et al. 2010 Czech Republic Certak Ectoparasites Various spp Dubska et al. 2009 England Central England Toxoplasma Sturnus vulgaris Peach et al. 1989 Spain Madrid Bacteria Columba livia Muñoz et al. 2010 Spain Murcia Fungus Columba livia Haro et al. 2005 Spain Santa Cruz Ectoparasites, Blood Columba livia Foronda et al. 2004 parasites, and Gastro- intestinal parasites France Besançon Bacteria Corvus frugilegus Boutterfroy et al. 1997 Germany Berlin Ectoparasites Columba livia Dautel et al. 1991 Iran Ahwaz Fungus Columba livia Zarrin et al. 2010 Italy Napoli Bacteria Columba livia Santaniello et al. 2007 Italy Bari Bacteria Columba livia Tarsitano et al. 2010 Japan Hokkaido Ectoparasites Various spp Iwasa et al. 1995 Malayasia Bangsar Bacteria and Gastro- Corvus spp Yong et al. 2008 intestinal Poland Cracow Ectoparasites Columba livia Rosciszewska et al. 2000 Portugal Lisboa Toxoplasma Columba livia Waap et al. 2008 Slovak Republic Košice Virus Columba livia Gronesová et al. 2009

Slovak Republic Bratislava Bacteria Columba livia Rehacek et al. 1984 The Netherlands Amsterdam Bacteria Columba livia Heddema et al. 2006 The Netherlands Amsterdam Fungus Columba livia Bart et al. 2008 USA Houston Bacteria and Gastro- Molothrus ater Johnson et al. 2004 intestinal USA Illinois Ectoparasites Cyanocitta cristata Mannelli et al. 1993 USA Detroit Gastro-intestinal Quiscalus quiscula Seneviratna et al. 1975 USA Urbana Bacteria Various spp Marrow et al. 2009 USA Boston Bacteria Branta canadensis Fox et al. 2006 USA Chicago Virus Various spp Loss et al. 2009 USA Various, especially New York, Virus Various spp., particularly McLean 2006 New Jersey, Connecticut Corvus brachyrhynchos USA Memphis Virus Passer domesticus McLean et al. 1983 USA New Jersey Toxoplasma Tyto alba and Columba livia Kirkpatrick et al. 1990 USA New Jersey Blood parasites Various spp Kirkpatrick and Sluthers 1988 USA New Jersey Ectoparasites Tyto alba Kirkpatrick and Colvin 1989 USA Fort Collins Bacteria Branta canadensis Kullas et al. 2002 USA New Jersey Bacteria Carpodacus mexicanus Hartup et al. 2001 USA Baltimore Bacteria Falco peregrinus Dement et al. 1986 USA Honolulu Blood parasites Various spp Shehata et al. 2001 USA Milwaukee, Grand Forks and East Gastro-intestinal Accipiter cooperi Rosenfield et al. 2009 Grand Forks USA Fort Collins Bacteria Columba livia Pedersen et al. 2006

Table 1.2. Summary of studies investigating spatial patterns of parasite-bird interactions.

Country City Parasite type Host Factors studied Summary of Results References Brazil Curitiva Ectoparasites Various spp Prevalence in urban A lesser prevalence of Arzúa et al. 2003 and natural places infested birds was found in the urban peripheral area than the natural sites in 17 host birds Brazil Palmas Blood parasites Various spp Prevalence and diversity Diversity and Belo et al. 2011 in urban and natural prevalence higher in areas the urban area France Dijon Ectoparasites Turdus merula Prevalence and intensity Tick infestation Gregoire et al. urban and rural significantly lower in 2002 urban areas Germany Munich Blood parasites Turdus merula Prevalence urban and Lower parasite Geue and rural prevalence in urban Partecke 2008 areas Germany Berlin Trichomonas Accipiter gentilis Trichomonas infectation High infestation in Krone et al. 2005 urban areas Germany Bonn Bacteria and No name provided Prevalence along an Generally, suburban Maetzel et al. Ectoparasites urbanisation gradient and urban sites with 2005 dense tree populations exhibited higher prevalence Japan Dijon Bacteria Turdus merula Prevalence urban and Weaker parasitism in Bentz et al. 2006 rural urban area

Luxembourg Luxembourg Ectoparasites No name provided Prevalence along an Positive correlation Reye et al. 2010 urbanisation gradient between the grade of urbanisation and infection of ticks, Paleartic region 33 countries Blood parasites Various spp. Richness Heterogeneously bird Møller et al. species had higher 2010 diversity USA Tucson Trichomonas Accipiter cooperi Prevalence urban and Prevalence of T. Boal et al. 1998 exurban gallinae was significantly greater in urban than natural nestling USA Atlanta Virus Various spp., Prevalence along an Prevalence increased Bradley et al. especially urbanisation gradient from rural to urban 2008 Cardinalis sites cardinalis USA Tucson and Ectoparasites Zenaida macrura Abundance comparation Birds in arid regions Moyer et al. Weslaco and Columbina in two urban areas with have fewer lice than 2002 inca arid and humid birds in humid region environments Western paleartic Tunis, Valencia, Ectoparasites and Turdus merula Prevalence and intensity Reductions in tick Evans et al. 2009 region (Tunisia, Madrid, Blood parasites in 11 paired urban and prevalence and Spain, The Groningen, rural populations intensity in urban Netherlands, UK, Sheffield, Berlin, areas. There are also Germany, Poland, Szczecin, large reductions in the Latvia, Czech Prague, Krakow, prevalence of avian Republic and Riga, Tallinn malaria in many, but Estonia) not all, urban areas

Table 1.3. Summary of studies on parasite effects on birds in urban centres.

Country City Parasite type Host Factors studied Summary of Results References Estonia Tartu Blood parasites Parus major Health impact Infected individuals were Ots and Hõrak heavier than uninfected 1998 ones in the urban area Estonia Tartu Blood parasites Parus major Coloration and parasite Plumage coloration Hõrak 2001 prevalence generally higher in rural birds. Reduction of coloration of urban birds could be the results of a greater exposure to infectious diseases France Paris Blood parasites Columba livia Immunity, parasite intensity Darker individuals had Jacquin et al. and melanin-based lower parasite intensity 2011 coloration and greater immune response Japan Gifu See Summary of Corvus macrorhynchos Evaluation of pathological It is not clear what Hirata et al. 2010 Results and Corvus corone conditions parasite caused pathological issues Spain Barcelona Blood parasites Columba livia Age-related differences in Higher intensity in young Sol et al. 2003 parasitism in urban areas birds USA Phoenix Blood parasites Pipilio aberti, Comparison between rural In general, urban birds Fokidis et al. Mimus polyglottos, and urban populations for had less blood 2008 Pipilo fuscus, blood parasite infection, parasitism. However, Toxostoma curvirostre, body condition (stored some birds exhibited and Passer domesticus energy reserves), and higher immune value immunity associated with probable stress or infection in urban areas

Improved rigour in methodology would facilitate measurements of other parasite groups as well. For example, visual examination is the predominant method for determining arthropod parasites, however other sensitive techniques, such as dusting, for collection and quantification are likely to improve the accuracy of prevalence estimates (Clayton and Walther 1997; see Chapter 2).

In contrast to the growing interest in studying factors that can influence the risk of parasitism inherent to the host, such as sex, age, and reproductive status, the number of publications about the habitat-specific factors that promote parasitism is comparatively small (Merilä et al. 1995; Kleindorfer et al. 2006), particularly for urban areas. One exception is the research carried out by Evans et al. (2009) who studied 11 paired urban and rural European populations of one of the most urbanised birds, Turdus merula.

Although they found a notable reduction of ticks in urban areas (with no ticks detected for 5 cities), the consistent and evident reduction was less conspicuous for blood parasites (Table 1.2). We need new studies carried out in other regions, for example in the Neotropics where urbanisation is chaotic (Biamonte et al. 2011) or in Australia where cities are more recent than in Europe.

Theoretical hypotheses about epidemiology suggest a positive relationship between abundance and/or prevalence of parasites and host density in some areas (e.g. Morand and Poulin 1998). Moreover, recent predictions suggest an increase of certain parasites

(e.g ticks, Kleindorfer et al. 2006) and pathogens in new environments as a consequence of climate change (Cumming and Van Vuuren 2006), thus bringing together the possibility of emerging diseases (Reye et al. 2010). Australia, Latin America and many

Oceanic islands are bioregions of major concern in this regard (Cumming and Van

Vuuren 2006). Bradley et al. (2008) found that antibody prevalence of the West Nile

Virus on birds increased from rural to urban sites. Similarly, the prevalence of

Trichomonas has expanded which has been associated with decline of some birds

(Lehikoinen et al. 2011). Supporting evidence has been published for some other vertebrates (Conner and Miller 2005).

Urbanisation can cause loss of host species diversity and an increase in the relative abundance of particular host species (McKinney 2008; Biamonte et al. 2011; Faeth et al.

2011). In cities, denser populations of some host species associated with overall low host diversity might mean that some host species are more susceptible to declines as a result of parasite infestation (Reisen et al. 2006; Koenig et al. 2010). Additionally, a number of studies documenting the effects of biodiversity in exposure, risk and transmission of diseases in ecological communities has found that a loss of host species diversity could change the infection rate between different hosts (Keesing et al. 2006).

However, since the patterns of diversity and abundance of hosts could vary between cities, generalisation about theories of disease risk and parasite exposure in urban areas and the presence of urban bird species might be difficult. For example parasite loads on a particular host are likely to be different in cities where they are abundant but associated with a poor alternative host diversity, compared to loads in cities with a lower population density of the particular host or in a city with a high diversity of other host species (Pongsiri et al. 2009; Keesing et al. 2010). This suggests that it might be difficult to discriminate between the effects of density and diversity. Therefore, to identify mechanisms involved in bird-parasite dynamics, studies should consider not just community structure and diversity of hosts, but also parasite dependent factors such as frequency of vectors, parasite identity, parasite parameters (e.g. prevalence,

43 intensity), parasite stages, potential interactions among different parasites, and specific traits (e.g. vector-borne or non vector-borne transmission).

Since bird hosts occurring in urban areas are interacting directly or indirectly with other individuals or species, and potential vectors, which can potentially affect parasite transmission and disease risk, cities with low diversity but a high abundance of urban species could experience an increase in diseases and pathogen prevalence due to the

Dilution Effect (Bradley and Altizer 2006), a phenomenon which explains why disease risk varies inversely with host diversity (Keesing et al. 2006). In brief, the Dilution

Effect describes a mechanism of increased disease risk and parasite incidence when host diversity is reduced. Thus when urban diversity is lower, transmission of diseases might be mediated by more frequent contact between hosts and infected vectors as a result of high density, followed by transmission to other susceptible hosts (as a result of increased contact due to high density), and a slow recovery from infection (Swaddle and

Calos 2008). Similarly, avian communal roost sites and perches (Harkinezhad et al.

2009), as well as human food sources (Jones and Reynolds 2008) could be implicated in the transmission of parasites and diseases (Ward et al. 2006; Diuk-Wasser et al. 2010).

Other hypotheses, such as the Enemy Release Hypothesis (success of exotic species due to elimination of parasites from their original habitat), the Novel Weapon Hypothesis

(susceptibility of native species due to exotic urban colonisers that bring new pathogens) (Marzal et al. 2011) and Amplification Effects (where biodiversity and disease risk are directly related) (Keesing et al. 2010) are valid frameworks which should be considered but remain to be studied in order to interpret how urban environments can affect parasite dynamics. As a result, we have very little understanding of whether parasites in cities could be an important constraint influencing

44 the species that can exist in these habitats.

While some bird species survive and prosper in urbanised areas, others are directly or indirectly affected by a wide range of anthropogenic activities and infrastructure (see

Evans et al. 2011). Birds in urban areas are exposed to an increase of some kinds of food and water, predation, competitive interactions, warmer temperatures and different types of pollution such as noise, artificial lighting and toxins (Chace and Walsh 2006).

Although some of these factors are potentially contributing to the inclusion or exclusion of birds in cities, they do not explain fully the presence, establishment and success of some native species that thrive in urban environments. It implies that other factors that are more cryptic might also be important.

Globally, I have found few studies to help elucidate the role of parasites in urban areas and the mechanisms that could influence the bird-parasite relationship. Although a reduction of parasite infestation was found as a potential factor favouring the presence of urban birds (Geue and Partecke 2008), species which are rare in urban environments might be more susceptible. Thus tolerant and sensitive urban birds might exhibit different susceptibility to parasite load thus influencing their ability to occupy urban centres. However, few studies have tested the relative importance of parasites and susceptibility in determining a species presence in urban areas (Geue and Partecke

2008). More comparative surveys (Evans et al. 2009; Evans 2010) using different parasite parameters between tolerant and urban sensitive birds and their conspecifics in adjacent natural areas will help to understand the dynamics of parasites and disease risk in urban centres.

The higher prevalence of diseases and parasites of birds recorded in some studies might be partially a result of a spillover from domestic and exotic animals (Lehrer et al. 2010).

Moreover, high population density of urban-adapted hosts and urban colonisation by exotic species could influence contact rates with, and among, host species and vectors and might favour the transmission of parasites (Keesing et al. 2006). Some are recognised as sources of potential diseases in humans (Polley 2005; Tsiodras et al.

2008), such as the West Nile Virus (Bradley et al. 2008) and Lyme disease (Maetzel et al. 2005). Additionally, pollutants deposited in urban areas could cause depressed immune systems increasing the impact of parasites (Combes 2001).

As migratory birds serve as reservoirs and/or mechanical vectors for numerous parasites, migratory species may play a significant role in the dispersion of pathogens

(Hubálek 2004; Alekseev 2006; Atkinson 2008; Tsiodras et al. 2008; Sehgal 2010) as has been recently confirmed for Trichomonas (Lawson et al. 2011). MacGregor-Fors et al. (2010) found that urban areas could offer winter habitat for some Neotropical migrant birds. If increasing urbanisation results in an increase in usage of cities by migratory birds, it could potentially cause a spread of parasites in cities and between natural and urban areas. Monitoring will tell more about the emergence of this potential issue.

While factors such as increased presence of exotic species or poorer quality of habitat

(Reye et al. 2010) might suggest an increase in parasitism in urban areas (Table 1.2), the situation is not conclusive with a range of studies showing lesser prevalence; for example, in Palaearctic (Evans et al. 2009) and Asian (Bentz et al. 2006) cities. Urban environmental conditions which alter diversity and abundance of vectors, some of

46 which could be essential for the transmission of parasites, could cause lower parasite abundance in urban areas (Bradley and Altizer 2006). The lack of vegetation in urban areas, as well as pesticide applications might inhibit the development of some ticks as suggested (but not tested) by Evans et al. (2009) (see also Evans 2010). Further, other authors have suggested that an increase in food availability probably helps urban birds through increased immunity (Fokidis et al. 2008).

The role of other factors, such as warmer microclimates apparently has differential effects on the parasite prevalence of bird parasites. For example, the urban heat island effect could increase the desiccation of ticks in urban areas (Evans et al. 2009), however, Alekseev (2006) suggested this could promote parasitism in cities. Warmer microclimates could make mosquitoes feed and reproduce faster (Githeko et al. 2000).

Parasites that persist in urban areas are apparently less known than those present in rural or natural areas (Mackerras and Mackerras 1960; McFarland 1986; Rojas et al. 1999;

Scheuerlein and Ricklefs 2004; Movila et al. 2008; Averis et al. 2009; Ogrzewalska et al. 2010). While, Chasar et al. (2009) explored the effect of degraded rainforest habitats on blood parasite diversity in African passerines, I found no study for passerines in urban areas in the African continent and very little information is available around the world. Additionally, studies on other parasites are few. Bacteria-bird interaction studies in urban areas are scarce and mainly focused in detecting negative effect groups (de

Sousa et al. 2010; de Lima et al 2011). Bacteria, however, can also be beneficial for the host (Moreno et al. 2003).

1.3.3. Parasite Effects on Host health

Even fewer studies investigated health and body condition in urban birds relative to parasite load. Papers were mostly published in Europe (Figure 1.1). Immunological and pathological conditions were used as health measures to detect responses potentially caused by blood parasite infestation (Table 1.3). For example, Fokidis et al. (2008) focused on blood parasites in Phoenix (USA), and showed how haemosporidians, body condition, and immunity covary in an urban versus rural setting in five passerines (Table

3). I found no paper focused on the effects of any other parasite types on health in urban areas.

If bird-parasite interactions are important in structuring avian communities in cities, then evidence is needed for urban sensitive species in conjunction with data for urban tolerant species. Future research should test if tolerant birds have less parasite infestation and better (or similar) health and body condition in cities than in natural areas, and if sensitive birds exhibit higher infestation by parasites in urban areas than natural areas which would also impact their health and body condition. Differences in parasite susceptibility may be related to different life history strategies (Atkinson et al.

2008) and might explain why birds with particular traits are more common in cities. For example, Jacquin et al. (2011) found that darker urban pigeons had lower levels of a blood parasite and their immune systems responded faster to infection. Møller et al.

(2010) showed that heterogeneously distributed bird species rarely colonised habitats as well as homogeneously distributed birds and that they were host to a greater diversity of blood parasites. Thus comparisons in parasite load between common and uncommon urban species should also be combined with comparisons amongst species with different

48 life history traits. Parasite load should also be linked with measures of health and stress in birds since parasites that do not influence fitness are unlikely to be factors in determining whether birds live in urban areas. However, an integrative approach among parasitism, immunology and physiology remains to be developed in urban areas.

Further, in Australia and the Neotropics, the bird fauna are derived from different evolutionary histories with many, in Australia, showing unusual cooperative breeding life histories which may well influence infestation (Oorebeek and Kleindorfer 2009).

Geue and Partecke (2008) suggested that the lower parasite prevalence could be one of the factors favouring the invasion of certain birds into urban ecosystems but studies are needed to help elucidate the role of parasites as a potential factor to maintain and regulate urban diversity (Hamer and McDonnell 2008). Finally, it will also be useful to understand the emergence and ecology of human and wildlife diseases as urban areas are where domestic animals and humans interact closely with wildlife (Bradley and

Altizer 2006).

Factors of stress such as noise and pollution, impact on the health of some vertebrates, including birds (e.g. Eens et al. 1999; Burger et al. 2004; Partecke et al. 2006; French et al. 2008). Such stresses may increase the likelihood of parasite infestations, affecting the capacity of hosts to occupy cities. In contrast, animals exhibit a variety of documented mechanisms to combat ectoparasites, including morphological, behavioural and immune responses (Boughton et al. 2011). Immune responses of animals in cities might explain the low prevalence of parasites in cities (Geue and Partecke 2008) for example in adult birds (Sol et al. 2003), and although self grooming is considered the first line of defence

(Hart 2000), grooming and immunological competence has not been addressed in the

49 urban context. Additionally, although there are a wide number of possible techniques available for immuno-ecology (Boughton et al. 2011), most studies have only used leukocyte profiles from microscopic slides (Davis et al. 2008). A range of other assays could complement our understanding of the link between stress, health and parasite loads.

Host immunity is also dependent on feeding habits (Sorci and Cornet 2010). In cities, for example, it is thought that higher immunity may be caused by a high abundance of food (Gregoire et al. 2002). Native parasites might have greater impact on introduced host species due to absence of appropriate immune response and/or resistance mechanisms (Dobson and May 1986). Similarly foreign parasites associated with exotic host species might spread to native hosts for the same reason (Freed et al. 2008).

Therefore, an integrative approach should explore food resources and parasite availability in cities and the effects on immune responses.

1.4. Conclusions

Parasite interactions have the potential to determine the distribution of their hosts, particularly where hosts are stressed on in poor quality environments. With cities growing globally, and urban diversity becoming important in the conservation of biodiversity, it is important to understand the role that parasites might have in limiting urban biodiversity. My review revealed a very large research gap in this area. In order to improve our understanding of host/parasite interactions in anthropogenic landscapes, a better understanding of the types of parasites, the mechanisms that influence their prevalence and their impact is needed.

CHAPTER TWO: EVALUATION OF A COMMERCIAL INSECTICIDE

POWDER FOR QUANTIFICATION OF BIRD ECTOPARASITES IN

ECOLOGICAL STUDIES

2.1. Introduction

Parasites are a dominant and diverse group which is fundamental in influencing ecological and evolutionary processes. Certain parasitic life strategies may allow the maintenance of ecological processes and will be critical to the conservation of fully functioning ecosystems in natural environments. For example, parasites can maintain species coexistence across space and time as they can have a detrimental effect on host fitness (Arostein et al. 2012). However, chronic alterations in parasite prevalence and introduction of parasites and pathogens to new environments might cause significant changes in vertebrate host populations and change ecological processes as has been recorded in remote islands (Samuel et al. 2011). Therefore, parasitism could contribute greatly to the structure, diversity, conservation, function and health of biodiversity.

In new environments, it is predicted that there will be an increase of certain parasites and pathogens which can increase the possibility of emerging diseases not experienced in natural undisturbed areas (Reye et al. 2010). Australia, Latin America and many

Oceanic islands are regions of major concern (Cumming and Van Vuuren 2006) due to changes in climatic conditions, habitat loss, fragmentation and a greater presence of human-modified landscapes. As a result, identifying and monitoring parasites is important to understand if the risk of parasitism and wildlife diseases caused by 51 ectoparasites increases due to human activity (Bradley and Altizer 2006).

A large diversity of arthropod ectoparasites has been recorded on birds (Clayton et al.

2010). They have detrimental effects on growth, fitness and survival of their bird hosts

(Moyer and Clayton 2004) and they could be vectors of a large variety of pathogens, which include viruses, bacteria and protozoa (Atkinson et al. 2008).

Ectoparasites such as lice (Phthiraptera), fleas (Siphonaptera), bugs (Hemiptera), flies

(Diptera), and mites and ticks (Acari) are known to occur on birds but data about their prevalence and their specific detrimental effects on hosts are still scarce. However, information of this type is especially important in tropical regions where parasite pressure is predicted to be greater (Poiani 1993; Cumming and Vuuren 2006) due to drastic environmental changes associated with urbanisation, habitat conversion, increased temperatures and fragmentation (McCallum and Dobson 1995; Daszak et al.

2000; Sutherst 2001; Bradley and Altizer 2006; Cumming and Vuuren 2006).

The scarcity of information on bird-ectoparasite interactions is partially compounded by the lack of powerful and comparable detection methods. Four ectoparasite collection methods on live birds are available in the literature (Clayton and Walther 1997): visual examination, anesthesia, trapping and dust-ruffling. For trapping, birds are restrained in cages and ectoparasites are sampled by placing the host in a cage over a pan of water into which the parasites can be collected, anesthesia using chloroform vapours (Poiani

1992) in fumigation chambers allow the bird to be examined once unconscious, although a safer design is to anaesthetise parasites of conscious birds using modified jars that do not expose the bird heads to the fumes. However, these chemicals are still

52 dangerous for researchers and birds (Clayton and Drown 2001). As a result, many bird- ectoparasite assessments have used visual examination on caught birds without restraint or anesthetic (McFarland 1986; Gregoire et al. 2002; Kleindorfer et al. 2006; Carrillo et al. 2007; Dubska et al. 2009; Evans et al. 2009; Oorebeek and Kleindorfer 2009). This technique usually samples a particular region of the body and there is an increased inaccuracy as observers use no magnification tool. While there is good comparative strength within each study, the ability to compare across studies will be hampered if different areas of the body are searched. Furthermore, overall assessments of prevalence may be badly underestimated, preventing broader generalizations being made.

Therefore, it has been suggested that other methods for quantification might offer a higher degree of accuracy in some parameters (Delgado-V. and French 2012) such as richness (number of taxa present), prevalence (proportion of infested birds in a population), and mean intensity (number of individuals of a particular parasite group in a sample of a host species divided by the number of infected individuals of the host species in the sample) (Clayton and Moore 1997).

Dust-ruffling with an insecticide represents a simple method that may improve estimates of parasite loads. Although, there are few field empirical data that quantitatively compare dust ruffling to visual inspection, studies have mostly been undertaken on a reduced number of species (e.g. the rock dove and the house sparrow) in temperate zones (Walther and Clayton 1997a, b) and/or for the screening of a particular group of arthropods (i.e. lice) on captive individuals (Clayton and Drown 2001).

Dust-ruffling methods have normally used products such as Pyrethrum (pyrethrin) and

Dri-Die (silicon dioxide) (Southwood and Henderson 2000) but there is no

53 documentation about the efficiency of other commercial products. Since Pyrethrum is not 100% effective and Dri-die has lethal side-effects on birds (Southwood &

Henderson 2000), an improved rigour in methodology using new commercial resources might facilitate measurements of ectoparasite quantification.

Pestene is an insect powder (Composition: Sulphur: 50 g/kg, Rotenone: 10 g/kg; Inca

Co. Pty Ltd, St Mary’s, Sydney, NSW, Australia) that is considered harmless for birds. It is commonly used in veterinary treatment and is becoming popular for control of lice, mites and fleas of domestic and laboratory animals (see Courtney Jones et al. 2012) but it has never been tested in ecological field studies. I compared dust-ruffing using

Pestene with the visual examination method for detection and quantification of ectoparasites on some Australian small passerines in order to determine if Pestene might be chosen as a quick, accurate and suitable methodological approach for ecological parasite studies.

2.2. Methods

Bird capture was undertaken at University of Wollongong, Wollongong, New South

Wales (34˚ 24’ 17” S 150˚52’ 15” E). Birds were mistnetted from February until May

2012. Once removed from the mist net, birds were individually transported in a clean cotton bag to the processing station where the ectoparasite methods were performed.

Each bird was released at the capture site immediately after dust-ruffling processing

(see below) and not resampled if caught again.

I conducted a visual inspection for any kind of ectoparasite arthropod by searching

54 head, throat, neck, under wings, around eyes, legs and belly for 3 minutes per bird by gently blowing feathers away. Additionally, I examined the right wing against a bright background in order to count parasites which are known to be between the barbs of the flight feathers of the wings and tail (e.g. feather mites). Parasites identified through visual examination were counted but not removed.

Immediately after the visual test, birds were dust-ruffled following Walther and Clayton

(1997) and Poiani et al. (2000) with some variations. I dusted each bird with ca. 2 grams of Pestene powder on rump, tail, wings, back and belly. The bird was held over a pan with a clear piece of paper as the collecting surface. Following dusting, the powder was distributed homogeneously all over the body for about 1 min through gentle rubbing with fingertips. The procedure was never done near face in order to avoid any potential irritating effect in the bird's eyes, however the use of Pestene is considered harmless for birds.

Birds were placed back in the cotton bag or in a paper bag and held for 10 min to allow the powder to kill parasites. Bags were big enough to allow preening and stretching out.

Afterwards, feathers all over the body were ruffled and ectoparasites were collected on the paper underneath. Ruffling was carried out for 1 min per bird. The collecting paper and holding bag were deposited in a clean ziplock plastic bag and assessed on the same day in the laboratory. Using a stereoscopic microscope (under 10x and 40x), ectoparasites were counted and collected from both the bag and the paper. Parasites were identified as ticks (Acari: Ixodida), lice (Insecta: Phthiraptera), feather mites

(Acari: Astigmata) or louse flies (Diptera: Hippoboscidae). Numbers of parasites found

55 by each method were compared using a paired t test. Prevalence was calculated as the number of birds caught with parasites while intensity was the number of individuals of a particular parasite group in a sample of a host divided by the number of infected individuals in the sample. Work was undertaken under UOW Ethics Number: AE11/20.

2.3. Results

Seventy-two individuals of 13 bird species were captured and sampled for ectoparasites using both methods: Red-browed finch [n = 32, Neochima temporalis], Superb-fairy- wren [n = 17 Malurus cyaneus], Silvereye [n = 6, Zosterops lateralis], Eastern yellow robin [n = 4, Eopsaltria australis], White-browed scrubwren [n = 4, Sericornis frontalis], Grey fantail [n = 3, Rhipidura fuliginosa], Golden whistler [n = 3,

Pachycephala pectoralis], Eastern spinebill [n = 1, Acanthorhynchus tenuirostris ],

Lewin's honeyeater [n =1, Meliphaga lewinii], Large-billed scrubwren [n = 1, Sericornis magnirostra], Yellow-throated scrubwren [n = 1, Sericornis citreogularis], Brown thornbill [n = 1, Acanthiza pusilla], and Bassian thrush [n = 1, Zoothera lunulata]). A total of 704 ectoparasites were counted by visual examination and 1718 by dust-ruffling

(Table 2.1).

Ectoparasites were mostly feather mites on all species (691 [Mean: 9.47 ± SD: 11.81,

Range: 0-47] by visual and 1521 [20.84 ± 20.91, 0-99] by dust-ruffling) (Table 2.1).

Lice were recorded on seven species but not on Eastern yellow robin, Eastern spinebill,

Lewin's honeyeater, Yellow-throated scrubwren, and Brown thornbill. Only 8.6% of lice were visually detected in advance (12 [0.4 ± 1.52, 0-8]) compared to 140 [4.67 ± 5.55,

0-27] recovered by the dust-ruffling method. Ticks (54 [3 ± 4.96, 0-23]) were only recovered using dust-ruffling on 6 species and were not found on Silvereye, Grey 56 fantail, Golden whistler, Large-billed scrubwren, Lewin's honeyeater, Brown thornbill, and Yellow-throated scrubwren. Four louse flies detected during the study were counted by visual examination on Superb-fairy wren, Red-browed finch and Eastern yellow robin, but one more was recovered by the use of Pestene dust-ruffling on the only individual of Bassian thrush.

Results show that the dust-ruffling method with Pestene is significantly more sensitive than the visual examination in recovering a greater number of feather mites and lice

(paired t-test mites; t60 = -5.49, p <0.001, lice; t29 = -5.30, p <0.001) (Table 2.1).

The proportion of infected birds (prevalence) varied between the methods. Furthermore, the estimates of infestation levels (intensity) also differed. Dust-ruffling was notably more sensitive for counting ectoparasites, identifying a greater proportion of the population that are infested by mites, lice and ticks and discerning a higher number of parasites per bird (Table 2.2). Contrary, louse fly counting was better through visual examination.

Table 2.1. Mean ± SD, range and total numbers of feather mites, lice, ticks and louse flies recovered from 72 passerines using visual examination and Pestene dust-ruffling methods.

Visual examination Pestene dust-ruffling Mites 9.59 ± 11.81, 0-47, 691 21.12 ± 20.91, 0-99, 1521 Lice 0.16 ± 1.52, 0-8, 12 1.94 ± 5.55, 0-27, 140 Ticks 0 0.75 ± 4.96, 0-23, 54 Flies 0.05 ± 0.71, 0-2, 4 0.01 ± 0.43, 0-1, 1

Table 2.2. Differences in prevalence and mean intensity of mites, lice, flies and ticks from a sample of 72 birds using visual examination and the Pestene dust-ruffling.

Prevalence Mean intensity Visual examination Mites 72.2% 13.3 Lice 4.2% 4 Flies 4.2% 1.3 Ticks 0% 0 Pestene dust-ruffling Mites 90% 25.8 Lice 41.6% 4.7 Flies 1.4% 1 Ticks 25% 3

2.4. Discussion

I recorded a clear disparity between the methods with dust-ruffling recovering more parasites. My results suggest that some previous research using the visual examination method (e.g. Gregoire et al. 2002; Evans et al. 2009) might have underestimated parasite loads. Accurate estimates of parasite loads are needed especially when relating parasites to the health and condition of birds and to understand declines (Ford et al.

2001; Valera et al. 2006; Delgado-V. and French 2012).

While visual examination is thought to be sensitive for the detection of ticks and lice, in our case it failed in detecting all ticks and only recorded a reduced fraction of lice that were recorded using Pestene. Visual examination has been employed for detection and counting of lice (Carrillo et al. 2007), however this technique has some limitation in 58 birds with dense plumage and small bodied lice (Clayton and Walther 1997), a problem

I encountered. Visual examination works well with big and permanent ectoparasites such as Ixodid hard ticks which are easy to see and count since they are normally imbedded on the host skin (Clayton and Walther 1997), particularly in naked areas.

However, tick quantification is difficult when they are small and immersed in dense plumage, especially on small passerines.

Contrary to more permanent ectoparasites, visual examination should be employed for the collection of louse flies because they leave the host quickly after capture, suggesting that examination for these should occur as soon as the bird is mistnetted (Poiani 1992;

Clayton and Moore 1997). However, caution should be exhibited when processing bigger birds by visual examination since small flies could be hidden on the plumage. In our case, one fly was undetected visually in a Bassian thrush remaining hidden until the dust-ruffling was conducted.

Based on my trial, I would suggest that Pestene could be used for ectoparasite studies to estimate levels of infestation. Additionally, intense visual searches for ectoparasites can cause eyestrain and headaches (Clayton and Moore 1997), and might be prone to error in detection when bird sampling is numerous and taken from small-sized birds. Despite the fact the visual examination could take less time compared on the dust-ruffling, to rely exclusively on the former method might undersample an important number and diversity of ectoparasites. However, since processing a bird that is caught often includes a visual exercise of measuring and evaluating condition for each bird, visual searches of ectoparasites can be partially done at the same time. This could help detect other ectoparasites that quickly abandon the captured hosts (such as fleas and louse flies) or

59 other static parasites which are deeply attached to the skin of the host (Clayton and

Walther 1997).

Estimates of feather mites might have been more similar between methods in the event that the visual screening had been carried out on both wings and tail (Clayton and

Moore 1997; Clayton and Drown 2001). However, the time spent in detecting and counting mites along all these parts could be similar to the time spent dust-ruffling with

Pestene, which might recover other ectoparasites.

2.5. Conclusions

Based in this brief trial, Pestene has a number of advantages that merits its use. It is a sensitive, practical, easy to perform, low cost technique for sampling a wide variety of ectoparasites. It is a quick method to quantify and provides a realistic estimate of prevalence and intensity. Its use is a valuable tool to explore the occurrence of ectoparasites on birds as well as the potential role of parasites in determining the structure, conservation and maintenance of bird communities, especially in small passerine birds.

CHAPTER THREE: URBANISATION DOES NOT INCREASE

ECTOPARASITE PREVALENCE IN AUSTRALIAN PASSERINE BIRDS

A modified version was submitted to be published in Urban Ecosystems:

Delgado-V., C.A., French, K., In review. Urbanisation does not increase ectoparasite prevalence in Australian passerine birds. Urban Ecosystems.

3.1. Introduction

Parasites are a dominant and diverse group which plays a fundamental role in ecological and evolutionary processes. Certain parasitic life strategies may allow ecological processes to be maintained and will be critical to the conservation of fully functioning ecosystems in natural environments (Samuel et al. 2011; Zylberberg et al.

2013). Parasites can maintain species coexistence across space and time as they affect host fitness, reduce dominance of host species and can additionally play important roles in determining species distributions (Nichols and Gómez 2011). However, chronic alterations in parasite prevalence and introduction of novel parasites and pathogens to environments might cause significant changes in host populations. High levels of protozoan parasites on bumblebees, for instance, can be fatal to both individuals and colonies (Goulson and Brown 2010).

In contrast to the growing interest in studying factors that can influence the risk of parasitism inherent to the host, such as sex, age, reproductive status and behaviour, the habitat-specific factors that promote parasitism have been mostly ignored (Kleindorfer 61 et al. 2006). However, human modifications to the environment, such as deforestation, can disturb the ecology of host-parasite relationships although particular consequences of growing global phenomena like urbanisation are largely unknown (Bradley and

Altizer 2006; Delgado-V. and French 2012). This is despite the fact that the risk of parasitism and wildlife diseases caused by ecto- and endoparasites are likely to increase with human activity (Bradley and Altizer 2006). Parasites and pathogens in conjunction with habitat degradation (caused for example by urbanisation) have been recently identified as major concerns in conservation biology as they may cause several detrimental effects in native species (Daszak et al. 2000; Cumming and Van

Vuuren 2006).

Urbanisation causes a loss of biodiversity in Australia and overseas (Gaston 2010), and might increase transmission of animal and plant infectious diseases (Keesing et al.

2010). Cities are also places of noise and pollution causing physiological stress which may increase the likelihood of parasitism (Pedersen et al. 2011). Urbanisation confronts animals with changes in habitat conversion and fragmentation. The reaction of animals to those disturbed and fragmented landscapes may affect the host parasite relationships in a complex fashion (Pedersen et al. 2011).

Theoretical arguments about epidemiology suggest a positive relationship between abundance and/or prevalence of parasites in urban areas (e.g. Morand and Poulin 1998;

Cumming and Van Vuuren 2006). However, the prevalence and impact of wildlife diseases and parasite loads in urban areas globally varies (Bradley and Altizer 2006;

Delgado-V. and French 2012; Brearley et al. 2013). Some authors have found that urban centres have lower rates of parasite infection than rural or natural areas in some groups

62 such as blowflies (Dykstra et al. 2012), ticks (Gregoire et al. 2002) and blood parasites

(Fokidis et al. 2008; Geue and Partecke 2008; Evans et al. 2009) not just in birds but also in other hosts (Page et al. 2008). In contrast, others have found that abundance and/or prevalence of parasites is higher in urban areas (Hammer et al. 2012; Kellner et al. 2012).

I studied natural and human-altered locations to test whether these habitats caused different levels of parasite infestation in small birds. I predicted that urbanisation caused a reduction in ectoparasite prevalence. This prediction has been corroborated in urban tolerant birds in Europe and South America (Gregoire et al. 2002; Arzúa et al. 2003;

Evans et al. 2009) but there is still no information about ectoparasite infestation dynamics in urban sensitive birds or other less urbanised birds.

Birds are the most prevalent vertebrate in cities and the best-studied taxonomic group.

As they are ubiquitous, they provide an excellent opportunity to study parasitism and its relation to urban development. However, most urban studies of bird parasites around the world have been conducted on a few urban-adapted species such as blackbirds, pigeons and house sparrows (Delgado-V. and French 2012). Here I investigate the spatial dynamics of ectoparasites in areas along an urbanisation gradient in two small passerines cataloged as urban sensitive birds in Australia (see Parsons et al. 2006).

3.2. Methods

I caught birds at five sites each along a gradient of urbanisation within and around

Wollongong, NSW, Australia (34° 26' S 150° 53'E). Using satellite images via Google

Earth (2014) analyzed using ArcGIS (10.2.1 version) ESRI (Redlands, CA, USA), we quantitatively determine the degree of urbanisation. Particularly, I calculated an approximate percentage cover of urban areas (buildings, infrastructure and sealed roads) surrounding study sites and used it to develop an Urbanisation Index (UI) as described in Gómez et al. 2008 and Hamer et al. 2012: UI = (100% – % green space + % impervious surface)/2 for the area within 1000 m of the sampling point. Surfaces considered as impervious included artificial structures such as asphalt, concrete, brick, and rooftops. Green space was defined as land having tree, shrub or grass cover.

The sites ranged from natural woodland to heavily urbanised areas. Sampling sites were: 1. Murramarang National Park (hereafter MNP, 35˚ 32’ 46” S 150˚ 21’ 49” E), a natural site situated ca. 300 km far from Wollongong to ensure the birds were well removed from urbanisation effects, UI = 1.05%; 2. Mount Keira (hereafter MK, 34˚ 24’

48” S 150˚ 51’ 19” E) was a sub-natural area located relatively close to urban development (i.e. 10k from Wollongong city), UI = 12.23%; 3. Ecological Research

Centre (hereafter ERC, 34˚ 24’ 17” S 150˚ 52’ 15” E) was a woodland edge surrounded by human altered habitats (e.g. urban gardens, paddocks and edge habitats), where there is a dense, shrubby understorey, UI = 28.12%; 4. Dalton Park (hereafter DP, 34˚ 23’ 47”

S 150˚ 53’ 55” E) was a city park surrounded by a matrix of house blocks with variable amounts of mowed grass and ornamental shrubs with occasional large trees, UI =

41.83%; and 5. Green House Park (hereafter GHP, 34˚ 26’ 31” S 150˚ 53’ 31” E), a small park situated between the industrial and commercial centre of the city which exhibits high traffic streets and scarce trees and bushes, UI = 48.41%. Sites were a minimum of 3 km apart located near the coast (within 0.5 – 4.9 km of the coast) where high infection has been found in Australia due to the wet environment (see Oorebeek

64 and Keindorfer 2009) (Figure 3.1).

Birds were caught using mist-nets erected from sunrise until noon during the breeding season 2012-2013 (from August to February). Nets were checked for captured birds every 10 to 15 min. Birds were examined for any signs of unusual stress upon capture in mist nets (e.g. panting, collapse, extreme shock molt), and any individuals judged to be in extreme distress was immediately released without processing.

Birds were individually transported in a clean cotton bag to the nearby processing station where they were sampled for ectoparasites. Birds were fitted with an aluminum leg band (supplied by the Australian Bird and Bat Banding Scheme) and released at the capture site immediately after processing. They were not included in the analysis if caught again. Birds were dust-ruffled following Walther and Clayton (1997) and Poiani et al. (2000) with some variations (Delgado-V. and French unpublished data, Chapter 2).

I dusted each bird with ca. 2 grams of Pestene powder on rump, tail, wings, back and belly. Pestene is an insect powder (Composition: Sulphur: 50 g/kg, Rotenone: 10 g/kg;

Inca Co. Pty Ltd, St Mary’s, Sydney, NSW, Australia) that is considered harmless for birds. It is commonly used in veterinary treatment and is becoming popular for control of lice, mites and fleas of domestic and laboratory animals (see Courtney Jones et al.

2012). The bird was held over a pan with a clear piece of paper as the collecting surface.

Following dusting, the powder was distributed homogeneously all over the body for about 1 min through gentle rubbing with fingertips. The procedure was never done near face in order to avoid any potential irritating effect in the bird's eyes, however the use of

Pestene is considered harmless for birds and is commonly used in veterinary treatment.

Birds were placed back in the cotton bag or in a paper bag and held for 10 min to allow

65 the powder to kill parasites. Bags were big enough to allow preening and stretching.

After this period, the feathers all over the body were ruffled for 1 min and ectoparasites were collected on the paper underneath. The collecting paper and holding bag were placed in a clean ziplock plastic bag and analyzed in the laboratory.

Using a stereoscopic microscope (under 10x and 40x), ectoparasites were counted from both the bag and the paper. Ectoparasites were determined as lice (Phthiraptera), ticks

(Ixoxida), and louse flies (Diptera: Hippoboscidae) which are ectoparasite groups which may cause direct (i.e. consumption of blood, skin, muscles, and other host's tissue) and indirect (i.e. associated adverse effects such as loss of weight) damage (Balashov 2007).

Prevalence was calculated as number of individuals of the host species infested with a particular parasite group divided by the number of hosts examined x 100 (Poiani 1992;

Oorebeek and Kleindorfer 2009).

A total of 314 individuals of 8 bird species were examined for ectoparasites: Red- browed finch [RBF (hereafter), n = 90], Superb-fairy-wren [SFW (hereafter), n = 84],

Silvereye (n = 3 Zosterops lateralis), Eastern yellow robin (n = 20, Eopsaltria australis), White-browed scrubwren (n = 46, Sericornis frontalis), Grey fantail (n = 1,

Rhipidura fuliginosa), Eastern spinebill (n = 43, Acanthorhynchus tenuirostris), Lewin's honeyeater (n =27, Meliphaga lewinii).

Data were analysed separately for each ectoparasite (lice, ticks and louse flies).

Logistic regression was used to examine the relationship between parasite infection

(presence-absence) and percentage of urbanisation. Individual analysis were undertaken for the Superb-fairy-wren Malurus cyaneus and Red-browed finch

Neochima temporalis only since they were the most captured species and occurred at all places sampled along the gradient.

3.3. Results

There was no significant effect of UI on the probability of lice infestation for both

2 2 species; RBF (χ1 = 3.101, P = 0.078) and SFW (χ1 = 1.224, P = 0.269) (Figure 3.2a).

However, the pattern of tick infestation varied between species. While increasing urbanisation resulted in a significant decline in the probability of tick infestation in RBF

2 2 (χ1 = 7.741, P = 0.005), there was a no clear effect in SFW (χ1 = 2.143, P = 0.143)

(Figure 3.2b). Finally, louse fly prevalence also changed significantly with degree of urbanisation. The probability of infestation decreased greatly in more urbanised areas in

2 2 both species (χ1 = 28.854, P < 0.0001, and χ1 = 12.225, P < 0.0005 for RBK and SFW, respectively) (Figure 3.2c).

Figure 3.1. Map displaying the ectoparasite sampling sites locations.

70 60 50 40 30 20 10

a. Proportion infested 0 0 102030405060 Urban degree

35 30 25 20 15 10 5

b. Proportion infested 0 0 102030405060 Urban degree

80 70 60 50 40 30 20 10 c. Proportion infested 0 0 102030405060 Urban degree

Figure 3.2 a. Lice, b. Ticks and c. Louse flies prevalences for Red-browed finch (■), and Superb-fairy wren (♦) along an urban gradient. Best fit lines for RBF (•••) and SFW (––) are depicted when the degree of urbanisation exhibited significant effects.

3.4. Discussion

Prevalence of parasites declined or remained unchanged with urbanisation, rather than increased. While lice were mostly more prevalent in urban fridge habitats (ERC), ticks and louse flies seem to be more sensitive to urbanisation since they were generally more prevalent in areas as urbanisation declined (MNP and MK) and less prevalent in highly urbanised areas (DP and GHP). My results suggest that the urban landscape may create unsustainable areas for ectoparasites which reduce their prevalence.

Although this is the first study to document the relationship between lice loads on birds and urbanisation (Delgado-V. and French 2012), some information is available on ticks and some groups of flies in urban areas. Higher prevalence of ticks in natural areas were also found in Brazil (Arzúa et al. 2003) and Europe (Gregoire et al. 2002;

Evans et al. 2009) which compared prevalence in natural, rural areas and ecotone sites with urbanised areas. Lower prevalence of ticks in more urbanised places might be due to higher temperatures, use of pesticides, unsuitable habitats, and variation or fewer intermediate and final hosts (Gregoire et al. 2002; Evans et al. 2009).

Although the hippoboscid flies have not been studied along urban gradients, other

Diptera groups vary in their response to urbanisation. Dykstra et al. (2012) report presence of blowflies (Calliphoridae) in rural areas but not in suburban landscapes, but botflies parasitism of Philornis (Muscidae) was more prevalent in residential areas

(Le Gros et al. 2011). My study found a clear decline in hippoboscid prevalence in more urbanised areas. As blowflies (Protocalliphora: Calliphoridae) appear to be affected by pollution (Eeva et al. 1994), the industrial proximities of my industrialised

70 site might affect the louse flies so they are less frequent compared with other areas.

Urbanisation can affect the presence of wildlife diseases positively and negatively

(Bradley and Altizer 2007) since parasite interaction with cities is probably highly dynamic, producing a pattern that includes specific responses based on the type of parasite linage and biology. One of the suggested factors that may be causing the dissimilar responses in these studies is that different species of birds may have different susceptibility to parasite loads. For instance, species which are common in cities may be advantaged by increased immunity whereas species which are now rare in urban environments may be more susceptible (Fokidis et al. 2008). According to this, it has been hypothesized that species sensitive (less frequent or declining) to urbanisation could exhibit a higher parasite infestation in urban areas than natural areas (Delgado-V. and French 2012). I found no evidence to support this hypothesis.

Both species I caught in adequate numbers were considered to be species that are affected negatively by urbanisation (Parsons et al. 2003). However, parasitism decreased in urban areas, rather than increased. This raises the possibility that hosts, as well as their parasite fauna, are negatively affected by urbanisation (Nichols and

Gómez 2011) regardless of the intrinsic differences in size, nesting and foraging behaviour that RBF and SFW exhibit. However, lice, ticks and louse flies are just a subset of parasites and other kinds of parasites could respond differently under disturbance due to urbanisation. For example, parasites and pathogens with simpler life cycles (such as gastrointestinal parasites) should be studied to detect whether they share common responses to urbanisation.

While lice are obligate parasites that complete their entire life cycle on the same host

71 and are transmitted by direct physical contact, other ectoparasites such as ticks have several development stages and require different species and substrates for survival and reproduction. Differences in their biology might explain differences in responses along the gradient. For example, while Page et al. (2008) reported a decrease in the intestinal parasite, Baylisascaris procyonis in racoons (the final host) with increasing urbanisation, Kellner et al. (2012) found that the prevalence of the same parasite exhibited an opposite trend on small mammals, the intermediate host of the parasite.

This suggests that a complete understanding of parasite dynamics requires the sampling of different hosts.

3.5. Conclusions

With so few studies on how parasites might be affected by urbanisation, this study makes an important preliminary contribution. For now, the overall pattern appears to be that urbanisation reduces ectoparasite prevalence in Australian passerine birds.

Evidence suggests that parasites can be found either at locations with intermediate development or they can even exhibit higher numbers in natural areas than those that are close to the city. Studies on different urban areas, seasons, parasites and a wider diversity of hosts are needed in order to understand the ecological distribution of parasitism in an urbanised world. In the future, sampling should also be undertaken in the non-breeding seasons of the hosts as parasites are known to change seasonally.

They may also interact with other parasite groups (c.f. Kleindorfer et al. 2006) which may influence colonization success when infestation by one parasite compounds the effect of another. Further as infection risk may differ among hosts, additional work is needed to identify how urbanisation influences specific bird/parasite interactions.

CHAPTER FOUR: DIFFERENTIAL INFLUENCE OF URBANISATION ON

COCCIDIAN INFECTION IN TWO PASSERINE BIRDS

A modified version of this chapter is published in Parasitology Research:

Delgado-V., C.A., French, K. In press. Differential influence of urbanisation on

Coccidian infection in two passerine birds. Parasitology Research. Research [DOI:

10.1007/s00436-015-4414-2]

4.1. Introduction

New cities are created every year and the already established ones expand in area and population (Evans et al. 2009), causing significant impacts on biodiversity through a range of processes. While the effects of urbanisation on some species have been well studied, interactions and relationships amongst species living in cities are virtually unknown (Delgado-V. and French 2012).

Parasite infection has been an important issue of study in natural ecosystem but parasite ecology in urban areas has not received enough attention yet. So that the extension of how urbanisation influences the prevalence and impact of parasites has not been studied in detail (Delgado-V. and French 2012), despite the fact that changes in host-parasite interactions have the potential to be one of the most important consequences of urbanisation (Bradley and Altizer 2007).

Birds are one of the most prevalent animal groups in urban areas. As they are ubiquitous in the urban environment even in highly urbanised areas, they facilitate the study of parasitism and its relation to urban development (Delgado-V. and French

2012). In addition, they are the most studied vertebrate in cities around the world so they offer further possibilities about parasite comparison among urban centres. Even though the same species are not found among cities, comparison might be conducted between biologies, guilts or behaviours.

Passerine birds are host to a variety of parasites which exhibit a diverse array of forms and life history, and where obligate and non-obligate taxa are recognised as vectors of pathogens and diseases. Passerine birds are mainly infected by Coccidia of the genus

Isospora (Apicomplexa: Coccidia), a cosmopolitan protozoan, which is transmitted via oocysts that are excreted along faeces of the host and infected by ingestion of sporulated oocysts (Dolnik et al. 2010). This is a group of parasites that can reduce weight, affect intestinal nutrient resorption, and reduce fertility (Atkinson et al. 2008).

Pathogens as Isospora parasites are capable of infecting multiple host species and some pose serious threats to human health and wildlife populations (Atkinson et al.

2008).

Comparative studies on parasites such as ectoparasites and blood parasites have been carried out among natural and urban sites (Delgado-V. and French 2012) but studies that had monitored changes in prevalence of other parasites along urban gradient are scarce (Reperant et al. 2007; Sitko and Zalésny 2014). For example, coccidians parasiting birds has kept virtually understudied in urban areas (Giraudeau et al. 2014)

74 despite the fact they are relatively easy and inexpensive to monitor.

Delgado-V. and French (submitted) documented a decrease of ticks and louse flies while lice kept unaffected by the degree of urbanisation. However, Evans et al. (2009) hypothesised that avian parasites and pathogens with more simple life cycles, such as some gastrointenstinal parasites, might exhibit a different response. This situation has been recently corroborated in the House finch (Haemorhous mexicanus), an urban tolerant bird (Giraudeau et al. 2014) in the US but there is still no information about

Coccidian infections in urban sensitive birds or other less urbanised birds.

The proposal of this paper is to document if other parasites may exhibit different responses to urbanisation that those recently found for ectoparasites (Delgado-V. and

French submitted). I tested the hypothesis that natural and human-altered locations present different levels of Isospora parasite infections on two passerine species which are both sensitive to urbanisation. Particularly, I explored the hypothesis that the overall Isospora infestation will be greater in areas with higher urban development.

Parasites are a diverse group which is fundamental in influencing ecological and evolutionary processes, therefore parasitism could contribute greatly to the structure, diversity, conservation, function, and health of biodiversity (Nichols and Gómez

2011). Knowing relative frequency of occurrence of these pathogens in the two species of urban-sensitive passerines with different biology and behaviour is essential for proper epidemiological studies, disease modelling and basic surveys that can subsequently be used as tools for species conservation programs in urban areas.

4.2. Methods

My study focused on two small passerine species known to be less abundant in urban areas (Parsons et al. 2006). Superb-fairy wrens Malurus cyaneus (SFW, hereafter) are small (14 cm), sedentary and territorial birds. The species lives in cooperatively breeding groups which usually occupy small (c. 13–24 ha) territories. SFW typically occur in various types of habitat with dense and shrubby understory, including grasslands with scattered shrubs, gardens and urban parks. SFW mainly eats insects

(Mulder 1995). Red-browed finch Neochima temporalis (RBF, hereafter) is a sociable small estrildid finch (11–12 cm long). It inhabits the east coast of Australia and is found in open and semi-open woodland. This species is sedentary or nomadic. RBF feeds mostly on seeding grasses but it also uses bird feeders. It is found in weedy areas along railway tracks, roads, creek lines and urban parks (Todd 1996).

I chose five sites that differed in their degree of urbanisation. The Urbanisation Index ranged from 1.05%-48.41% based on Gómez et al. (2008) and Hamer et al. (2012) index

(described in Chapter 3).

Field work was conducted over a year in breeding and non-breeding seasons. Birds were sampled from February 2012 to March 2013 using mist-nets. Nets were checked for captured birds every 10 to 15 min. Birds were examined for signs of unusual stress upon capture in mist nets (e.g. panting, collapse, extreme shock moult), and any individuals judged to be in extreme distress was immediately released without processing. Birds were individually transported in a clean cotton bag to the nearby processing station where a faecal sample was collected over a twenty minute period. Birds were fitted with

76 an aluminum leg band (supplied by the Australian Bird and Bat Banding Scheme) and released at the capture site immediately after processing and not included in the analysis if caught again.

Fresh excreta were collected in small vials containing 2.5% potassium dichromate

(K2Cr2O7) water solution following Dolnik (2006). The samples were stored at room temperature for at least 4 days until analysis. Oocysts of Isopora were extracted using the method described in Dolnik (2006). Oocysts were then transferred to a slide and examined under the microscope where I recorded the presence of oocysts. The prevalence of Isospora was determined as the percentage of birds infected at each study site.

Data were analysed separately for each host. Logistic regression was used to examine the effects of the degree of urbanisation and season (breeding and non bredding) on parasite prevalence. Interactions were included in the initial models and model simplification carried out by stepwise removal of explanatory factors that did not contribute significantly.

4.3. Results

I sampled 278 birds; 138 SFW and 140 RBF. Overall, there were 55 SFW infected

(19.78%) and 66 RBF (47.14%). RBF had a higher prevalence of Isospora than SFW (χ2

= 18.201, df = 9, P = 0.0329).

2 In RBF, the prevalence of Isospora increased significantly in more urbanised areas (χ1

= 4.09, P < 0.005) (Figure 4.1a). Prevalence did not change between breeding and non-

2 breeding seasons (χ1 = 3.16, P = 0.075) but there was a significant interaction between

2 the two (χ1 = 12.09, P = 0.005) (Figure 4.1a). In contrast, in SFW neither degree of

2 2 urbanisation (χ1 = 0.06, P = 0.8010) nor season exhibited significant effects (χ1 = 0.03,

P = 0.9555) on the prevalence of coccidians (Figure 4.1b).

4.4. Discussion

In this study, I tested whether the degree of urbanisation affects the occurrence of

Coccidian infections in two urban sensitive birds. I found that prevalence was differential between species, and that, contrary to the prediction, prevalence of

Isospora increased in more urbanised areas on Red-browed finch, in both breeding and non-breeding seasons, but did not significantly change with degree of urbanisation in Superb-fairy wren. Diet, behaviour, and habits, are suspected to be more influential factors on the variation observed between both species, where granivorous and gregarious species being significantly infected.

This study provides the first quantitative baseline information of Isospora prevalence along an urban gradient in Oceania. It shows that Coccidians can respond differently depending on the host suggesting that these parasites might be more prevalent in urban areas in some birds but not all. In one species, parasite prevalence in the population increased with urbanisation suggesting that fitness may be affected in birds in more highly urbanised areas. These differences of parasite prevalence may be link to other life history features which need to be further explored.

The prevalence and intensity of infection with Coccidian parasites in wild passerines is highly variable amongst species. In natural areas, Zinke et al. (2004) and Dolnik et al. (2010) found that ground feeder birds have the highest prevalence and intensity of infection, and that these parameters have lower values in insectivore passerines. Also, foraging in flocks may cause an aggregation of faeces at feeding sites, so that social foragers appear to be more exposed to coccidian infections (Giraudeau et al. 2014).

Additionally, infection may also occur during bathing, collection of nest material or pairing where flocks facilitate the transmission of parasites and lead to higher intensities of Coccidians. Many birds foraging at the same area for extended periods of time can result in accumulation of faeces and posterior transmission (Zinke et al.

2004; Dolnik et al. 2010).

My results contrast to other studies which have shown reduced parasite load in urban areas (Gregoire et al. 2002). For example, Geue and Partecke (2008) and Evans et al.

(2009) worked on the Eurasian blackbird Turdus merula and found a decrease in tick prevalence and intensity in cities. Sitko and Zalésny (2014) found a reduction in the helminth fauna in Eurasian blackbird in urban areas compared with forested sites.

Fokidis et al. (2008) found that some urban passerines were less affected by haemoparasites than its rural counterparts in the US desert.

As life histories of parasites vary extensively, responses to urbanisation may also differ. Prevalence and intensity of avian parasites and pathogens with simpler life cycles might be higher in urban areas (Evans et al. 2009) as transmission is easier.

Drastic environmental changes associated with urbanisation, habitat conversion, increased temperatures and fragmentation (McCallum and Dobson 1995; Daszak et al.

2000; Bradley and Altizer 2006; Cumming and Vuuren 2006; Calegaro-Marques and

Amato 2014) might cause declines in microhabitats, water availability and the abundance of intermediate hosts and vectors which are likely to affect ticks, helminths and blood parasites with complex life histories (Reperant et al. 2007; Evans et al.

2009; Sitko and Zalésny 2014). However, Isospora and other parasites, viruses and pathogens might have a different response as transmission pathways are quite distinct.

Isospora infections are passed between hosts by faecal-oral transmission where infective oocysts can contaminate the external environment, including food and water

(Dolnik 2006; Dolnik et al. 2010). This suggests that urban aggregations of host species may encourage the spread of parasites that are transmitted by direct contact.

For example, Hamer et al. (2012) found the avian exposure to West Nile Virus increased with level of urbanisation. Similarly, Giraudeau et al. (2014) recorded an increase of prevalence of poxvirus associated with urbanisation degree.

My results only partially support this hypothesis as prevalence was only related to urbanisation in one species, not both. Therefore, factors of exposure to parasites, such as diet, behaviour, and environmental factors, need to be investigated to explain the variation seen between RBF and SFW. For example, larger communal roosts or reduced areas for foraging and feeders might provide more transmission opportunities

(Giraudeau et al. 2014) facilitating contact rates and potential dispersal and transmission.

Differences in diet may also influence transmission rates. Dolnik et al. (2010) suggested that insects are less likely to be contaminated with sporulated oocysts than berries or seeds, resulting in both a lower probability of exposure and a lower dose for insectivores in comparison with granivores. Ground foraging granivores might be particularly susceptible to infection where birds may encounter faecal material.

Granivorous passerines, such as RBF might represent the most vulnerable birds under an eventual chronic prevalence of Isospora in urban areas (Giraudeau et al. 2014).

Alternative routes of uptake of transmission of oocysts might also explain differences

81 in seasonal prevalence. During breeding seasons, nestlings and siblings can be infected by parents during ingestion of contaminated food (Dolnik et al. 2010), causing a higher prevalence of Isospora in the breeding season in RBF along the sampled gradient of urbanisation.

4.5. Conclusions

Urban areas are becoming important areas for conserving and managing urban wildlife (Kantsa et al. 2013). Parasites could contribute greatly to the structure, diversity, conservation, function, and health of urban biodiversity, and future studies focusing on the understanding of the transmission dynamics and the ecology of wildlife pathogens in species will corroborate if this pattern of Isospora infection in granivorous and insectivore passerines can be applied more generally.

CHAPTER FIVE: PREVALENCE AND LINEAGE DIVERSITY OF AVIAN

HAEMOSPORIDIANS FROM URBAN AND NATURAL AREAS IN EASTERN

AUSTRALIA

5.1. Introduction

Parasites are a dominant and diverse group which is fundamental in influencing ecological and evolutionary processes. Haemosporidian parasites (Haemosporidia,

Apicomplexa) of avian hosts may play important roles in sexual selection and reproductive success in birds (McCallum and Dobson 1995; Atkinson 2008) and are recognised as factors that cause morbidity and mortality in avian species (Palinauskas et al. 2008). However, their pathogenicity is related to factors such as host nutrition, host availability, susceptibility and environmental stress. Additionally, a number of studies documenting the effects of biodiversity on exposure, risk and transmission of diseases in ecological communities have found that a loss of host species diversity could change the infection rate between different hosts (Keesing et al. 2006).

Haemosporidians have been recorded on a wide variety of vertebrates (Santiago-

Alarcón et al. 2014) where the most commonly occurring haematozoa in birds are species of Haemoproteus, Leucocytozoon and Plasmodium which are transmitted by a wide range of vectors. Avian Haemosporidians have a broad geographic distribution (Santiago-Alarcón et al. 2012) and are known for causing different impacts on individuals, populations and communities. For instance, avian

Haemosporidians may have significant impacts on fitness and reproduction of hosts which may cause decline and extinction of certain species as recorded for some 83

Hawaiian passerines (Atkinson et al. 1995).

Australian avifauna has a vast number of endemic species with different life- history characteristics associated with a unique evolutionary history with a long period of isolation. Additionally, several avian parasite groups of Australia may well be equally evolutionarily distinct and show unique lineages with different patterns of prevalence to other continents (Clark et al. 2014). However, little is known about bird-parasite relationships and whether they are composed of unique or endemic partners and how they are affected by human-induced changes such as urbanisation. The east coast of the country, a region that has experienced an expanding urban development in recent years, is a critical place where bird-parasite relationships may be changing.

Urban areas are increasing in both extent and number (Evans et al. 2009) which is causing significant impacts on loss of biodiversity. While the effects of urbanisation on biodiversity structure and composition have been well studied, interactions and relationships amongst species are virtually unknown. In new disturbed and anthropogenic environments, such as urban areas, it is predicted that there will be an increase in parasites and pathogens as hosts become susceptible under stressful changes in habitats, as well as from the increased possibility of emerging diseases not experienced in natural undisturbed areas (Reye et al. 2010). Australia, Latin America and many Oceanic islands are regions of major concern (Cumming and Van Vuuren

2006) due to changes in climatic conditions, habitat loss, habitat conversion, increased temperature, fragmentation and a greater presence of human-modified landscapes

(McCallum and Dobson 1995; Daszak et al. 2000; Sutherst 2001; Bradley and Altizer

2006; Cumming and Van Vuuren 2006; Garamszegi et al. 2011; Loiseau et al. 2013).

Identifying and monitoring parasites is important to understand if the risk of parasitism and wildlife diseases caused by parasites increases due to human activity

(Bradley and Altizer 2006). However, in contrast to the growing interest in studying factors that can influence the risk of parasitism inherent to the host, such as sex, age, and reproductive status, the number of publications about the habitat-specific factors that promote parasitism is comparatively small (Merilä et al. 1995; Kleindorfer et al.

2006), particularly for urban areas. A recent review (Delgado-V and French 2012), identified a gap in the literature of bird-parasite interactions in urban areas. Studies identifying parasites are more common for American and European urban birds where the parasite fauna is better known, although data is restricted to few bird hosts from a few cities (Delgado-V and French 2012). Understanding of parasite-host relationships has important implications for conservation and management of urban biodiversity, therefore it is crucial to determine which parasites survive in urban areas especially where urbanisation has been increasing (Jansen et al. 2009). Additional studies are urgently needed to increase our understanding of the diversity, prevalence, and distribution of Haemosporidians.

The objectives of the present study are 1. to analyse the prevalence and diversity of Haemosporidian parasites infecting birds in an urban environment of NSW,

Australia; 2. to compare the lineage diversity and Haemosporidian prevalence of

Plasmodium, Haemoproteus and Leucocytozoon between hosts inhabiting natural

85 and urban areas in order to detect the influence of urbanisation and, 3. to analyse the results obtained in this study in relation to other areas of Australia and the

Southern Hemisphere based on a compilation of references about haemosporidian prevalence. Several excellent literature analyses have been published recently that have compiled research largely from temperate natural areas of the Northern hemisphere (Garamszegi 2011; Lacorte et al. 2013; Clark et al. 2014). As a result my comparison was restricted to 1. natural areas from the Southern Hemisphere, and 2. urban studies all over the world. This decision allowed me to focus my comparison of the situation in natural and disturbed areas of the Southern

Hemisphere which is one of regions less explored about its parasite fauna

(Delgado-V. and French 2012; Clark et al. 2014).

5.2. Methods

5.2.1. Field sites

The study was conducted from March 2012 to February 2013 in four sites in

Wollongong and nearby areas, New South Wales, Australia. Birds were sampled at three sites in urban areas located in the city of Wollongong: 1. Ecological Research Centre at

University of Wollongong (hereafter ERC, 34˚ 24’ 17” S 150˚ 52’ 15” E), an urban- fringe area characterised by a woodland edge surrounded by human altered habitats (e.g. urban gardens, edge habitats), where there is a dense, shrubby understorey; 2. Dalton

Park (hereafter DP, 34˚ 23’ 47” S 150˚ 53’ 55” E), a city park surrounded by a matrix of house blocks. This park is located at a mosaic of roads, houses and yards with variable amounts of moved grass and ornamental shrubs with occasional large trees; and 3.

Green House Park (hereafter GHP, 34˚ 26’ 31” S 150˚ 53’ 31” E), a small park situated between the industrial and commercial centre of the city surrounded by high traffic streets with scarce trees and bushes. One natural area was also sampled; Murramarang

National Park (35˚ 32’ 46” S 150˚ 21’ 49” E) is situated ca. 300 km south of

Wollongong and well away from urban expansion.

5.2.2. Bird capture and handling

Birds were caught using mist-nets erected from sunrise until noon. Blood samples were collected from the brachial vein. A drop of blood was immediately placed on a clean glass microscopic slide to prepare a blood smear for microscopic examination.

Smears were air-dried in the field and stored until fixation and differential stain using

Kwik-Diff stain (Thermo Scientific™) as suggested by the manufacturer. Small dot samples of blood was collected and preserved on Whatman FTA Cards for molecular analysis.

5.2.3. Analysis and measurements of blood parasite infection by microscopy

Blood parasites were detected and determined by microscopic examination (Clark et al.

2009). Blood parasite prevalence was determined by surveying fixed blood smears on microscope slides. Smears were studied using light microscopy at high magnification

(100x) under oil immersion and 80 randomly chosen fields with homogeneous distribution of red blood cells were scored for presence/absence of blood parasites.

When no parasites were detected after that number of fields, the individual was considered uninfected.

A total of 270 individuals of 17 bird species (representing 10 families) were sampled for blood parasites using blood smears (Table 5.1). All samples were obtained from urban sites (i.e. from ERC, DP and GHP) and no slides were available from the natural site.

5.2.4. Molecular detection

Blood samples from 147 individuals from three passerine host species [the Eastern yellow robin (Eopsaltria australis) (n=8, 6 natural and 2 urban), the Superb-fairy wren

(Malurus cyaneus) (n=74, 46 natural and 28 urban), and the Red-browed finch

(Neochmia temporalis) (n=65, 45 natural and 20 urban)] were screened using molecular methods. Parasite diversity and prevalence were analysed separately for each host and compared between areas.

DNA was extracted using the Phenol-chloroform method following Sambrook and

Russell (2001) with some minor modifications. Nested-polymerase chain reaction

(PCR) protocol developed by Hellgren et al. (2004) was used which enables simultaneous detection from the three most common avian blood parasite genera:

Haemoproteus, Plasmodium, and Leucocytozoon. The screening method consists of an initial general PCR that allows the amplification of the three genera (primers

HaemNFI/HaemNR3). The second PCR amplified Plasmodium/Haemoproteus (primers

HaemF/HaemR2), and the final one, Leucocytozoon (primers HaemFL/HaemR2L).

Positive and negative individuals were identified by the presence or absence of a ~500 bp band on 2% agarose gels stained with GelRed, and visualized with a UV light source.

Further identification was completed by the sequencing of a fragment of 480-bp of the

88 cytochrome b following Bensch et al. (2000) and Hellgren et al. (2004). Obtained sequences were aligned and edited using the program Geneious v.8.0.2

(http://www.geneious.com, Kearse et al. 2012). DNA sequences were compared for identification to their closest matches in MalAvi (Bensch et al. 2009) and GenBank.

Natural and urban parasite prevalences for each host species were compared by Fisher

Exact Test using Quantitative Parasitology (Reiczigel and Rózsa 2005).

5.2.5. Literature review

For comparison to field data obtained on this thesis with other studies in Australia and overseas, a multi-keyword field literature search including the terms “haemoparasite”,

“prevalence”, “parasite” and “passerine” was performed in the databases PubMed, Web of Science, Biological Abstracts, and Summon. Also additional articles were reviewed from initial identified publications in order to expand my literature dataset. Research carried out on introduced, pets, and poultry were excluded from analysis.

Specific data such as geographic locality (tropical or temperate zone, hemisphere, continent, country and natural, rural or urban site), method of sampling, years of survey period, type of Haemosporidian, prevalence, type of bird, number of species and individuals sampled which were infected were extracted.

5.3. Results

5.3.1. Overall urban Haemosporidian prevalence by microscopy screening

Using visual inspection of microscope slides, I only detected two individuals with

Haemoparasites. It was not possible to determine the species of any blood parasite due to the low levels of parasitaemia (only one parasite per smear was detected through the screening), however, I was able to assign a genus. Both individuals (from two different species, the Silvereye Zosterops lateralis and the Eastern spinebill Acanthorhynchus tenuirostris) were infected with Haemoproteus (Table 5.1). I did not detect any individual infected with Leucocytozoon or Plasmodium.

5.3.2. Comparison of prevalence and lineage diversity between urban and natural areas using sequencing

From 147 screened birds, 26 individuals (17.7%) were positive for either

Plasmodium/Haemoproteus or Leucocytozoon infections. Irrespective of host species, total prevalence of Plasmodium/Haemoproteus was 2% and the prevalence of

Leucocytozoon was 15.7%. No Haemosporidian infection was detected in Eastern yellow robin but mixed infections were found in both Red-browed finch and Super-fairy wren (Table 5.2). Overall prevalence was significantly higher in the natural (23.7%) than urban areas (6%) (p<0.05). However, whereas Leucocytozoon occurrred in both areas, Plasmodium/Haemoproteus infection was only found in natural areas.

For Superb-fairy wren, individuals from natural areas exhibited higher total prevalence 90 of Haemosporidians (21.7%) than urban individuals (7.1%). Similarly,

Plasmodium/Haemoproteus as well as Leucocytozoon infection in SFW were both higher in natural areas (Table 5.2) but differences were not significant in any case

(p<0.05). RBF had a higher prevalence of parasites in natural areas (Table 5.2) and both total Haemosporidians and Leucocytozoon were more prevalent in natural areas

(F=0.047, p=0.027, and F=0.05, p=0.03 respectively).

DNA of 14 individuals was sequenced and four mitochondrial lineages were found which have not been reported in previous studies and are described for the first time here. The new lineages recorded are two Leucocytozoon lineages (referred to as lineages

MALCYA01 and MALCYA02 hereafter) and two Haemoproteus lineages (MALCYA03 and MALCYA04) which matched with no previous sequences available in MalAvi or

GenBank (Table 5.3). While MALCYA02 lineages were presented in SFW and RBF hosts, MALCYA01, MALCYA03 and MALCYA04 lineages occurred exclusively in

SFW individuals (Table 5.4). Parasite diversity was higher in the natural area than in the urban area. All four lineages were found in natural areas and just one (MALCYA02) was also found in urban areas (Table 5.4).

5.3.3. Literature review

I gathered 26 papers dealing with Southern Hemisphere regions and/or global urban areas. I found 20 papers with 50 estimates of parasite prevalence for natural areas (Table

5.5) and nine papers with 19 prevalence estimates for urban areas (Table 5.6). For natural areas, tropical region from Southern Hemisphere have a higher average prevalence of overall Haemosporidians (17.8%) than temperate region (14.9%) (Table

5.5). Similar patterns are exhibited by particular parasites. For instance, Haemoproteus prevalence is higher in tropical zones than temperate areas (22.2% vs 9.2% respectively).

There are differences in prevalence among continents for natural areas. Africa has the highest average prevalence of Haemosporidians (58.5%) followed by South America

(19%) and Oceania (15%). A similar pattern is exhibited by particular parasites such as

Plasmodium which has 58.5%, 16% and 8.8% prevalence from Africa, South America and Oceania in natural areas. Overall, however, Australia appeared to have lower levels of parasitism than all continents.

Cities located in the Southern Hemisphere have similar prevalence of Haemosporidians

(16.6%) to cities from Northern hemisphere (16.4%) (Table 5.6). There are some differences among urban centres on different continents. European (33.1%) and South

American cities (31.4%) appeared to have higher parasite prevalence than Oceania

(11.6%), North American (9.8%) and Asian cities (3.1%). The least frequent

Haemosporidian in urban areas is Leucocytozoon. This parasite was detected only in four of the 19 studies conducted. Leucocytozoon occur in European (49%), Oceanian

(22.05%), and Asian cities (4.6%). Plasmodium and Haemoproteus were the most common parasites, reaching their highest prevalences in urban areas from Europe and

North America particularly (Table 5.6).

Based on analysis of literature, I detected that average prevalence in Australia is higher in natural (17.8%) (Table 5.5) than urban areas (11.6%) but compared to other cities tended to be lower (Table 5.6).

Family Bird species N I L H P Acanthizidae Brown thornbill - Acanthiza pusilla 1 0 Large-billed scrubwren - Sericornis magnirostra 1 0 White-browed scrubwren - Sericornis frontalis 37 0 Yellow-throated scrubwren - Sericornis citreogularis 2 0 Estrildidae Red-browed finch - Neochmia temporalis 70 0 Maluridae Superb-fairy wren - Malurus cyaneus 69 0 Meliphagidae New Holland honeyeater - Phylidonyris novaehollandiae 1 0 Eastern spinebill - Acanthorhynchus tenuirostris 35 1 (2.86%) x Lewin's honeyeater - Meliphaga lewinii 13 0 Monarchidae Satin flycatcher - Myiagra cyanoleuca 1 0 Muscicapidae Grey fantail - Rhipidura albiscapa 3 0 Willie wagtail - Rhipidura leucophrys 6 0 Bassian thrush - Zoothera lunulata 1 0 Pachycephalidae Golden whistler - Pachycephala pectoralis 4 0 Pardalotidae Spotted pardalote - Pardalotus punctatus 1 0 Petroicidae Eastern yellow robin - Eopsaltria australis 16 0 Zosteropidae Silvereye - Zosterops lateralis 9 1 (11.11%) x Total 270 2 (0.7%)

Table 5.2. Prevalence (% of birds with infection) of Haemosporidians with no specific parasite discrimination (TH), Plasmodium/Haemoproteus (P/H), and Leucocytozoon (L) infections for Superb-fairy wrens and Red-browed finches in urban and natural sampling sites.

Sampling area TH P/H L Superb-fairy wren Urban area (n=28) 7.1 0 7.1 Natural area (n=46) 21.7 4.3 17.4 Red-browed finch Urban area (n=20) 5* 0 5* Natural area (n=45) 28.9* 2.2 26.7*

Table 5.3. MalAvi accession numbers of lineages identified in this study.

Parasite Lineage MalAvi Accession number Leucocytozoon sp. MALCYA01 To be determined Leucocytozoon sp. MALCYA02 To be determined Haemoproteus sp. MALCYA03 To be determined Haemoproteus sp. MALCYA04 To be determined

Table 5.4. Area and number of individuals of Superb-fairy wren (SFW) and Red- browed finch (RBF) infected by each mitochondrial lineage of Haemoproteus and Leucocytozoon parasite.

Host Area Lineages MALCYA01 MALCYA02 MALCYA03 MALCYA04 Leucocytozoon Leucocytozoon Haemoproteus Haemoproteus sp. sp. sp. sp. SFW Natural 1 3 1 3 Urban - 1 - - RBF Natural - 5 - - Urban - - - - Total 1 9 1 3

Table 5.5. Haemosporidians prevalence data obtained for natural sites located in the Southern Hemisphere recovered from the literature review. Abbreviations include: Type of Haemosporidian (P: Plasmodium, H: Haemoproteus, L: Leucocytozoon) and P (Prevalence in %).

Continent Country Date Haemosporidian Type of bird N sampled P (%) Reference

MICROSCOPY

Tropical

Africa South Africa 2004 P Passerines 104 58.5 Garamszegi 2011

Oceania American Samoa 1996 P Passerines 214 0.0 Garamszegi 2011

Oceania American Samoa 2001-2005 P Passerines 857 1.0 Atkinson et al. 2006

Oceania Australia 1975 P, H Non passerines 316 7.0 Bennett et al. 1975

Oceania Australia 1997-2001 L Passerines & 3059 51.1 Adlar et al. 2004 non passerines

Oceania Australia 1997-2001 H Passerines & 3059 31.4 Adlar et al. 2004 non passerines

Oceania Australia 1997-2001 H Passerines & 3059 10.9 Adlar et al. 2004 non passerines

South America Bolivia 1988 P Passerines 640 0.8 Garamszegi 2011

South America Bolivia 1988 P, H Passerines & 641 5.1 Benett et al. 1991 non passerines

South America Brazil 2000-2001 P, H Passerines 436 17.7 Sebaio et al. 2010

South America Brazil 2000-2001 P, H Passerines 489 13.9 Sebaio et al. 2010

South America Brazil 2000-2001 P Passerines 925 9.2 Sebaio et al. 2010

South America Brazil 2000-2001 H Passerines 925 3.2 Sebaio et al. 2010

South America Brazil 2000 P Passerines 246 23.2 Garamszegi 2011

South America Brazil 2005-2009 H Passerines & 772 7.1 Fecchio et al. 2011 non passerines

South America Brazil 2005-2009 P Passerines & 772 3.6 Fecchio et al. 2011 non passerines

South America Colombia 1999 P Passerines 159 0.4 Garamszegi 2011

South America Colombia 1999 P Passerines 315 1.1 Garamszegi 2011

Temperate

Oceania Australia 2004 H Passerines 110 9.1 Kleindorfer et al. 2006

MOLECULAR

Tropical

Oceania Australia 2002, 2003 H Passerines 219 28.0 Beadell et al. 2004

Oceania Australia 2002, 2003 P Passerines 219 14.0 Beadell et al. 2004

Oceania Australia 2005-2006 P, L, H Passerines 403 32.3 Zamora-Vilchis et al. 2012

Oceania Australia 2005-2006 H Passerines 403 19.9 Zamora-Vilchis et al. 2012

Oceania Australia 2005-2006 L Passerines 403 6.2 Zamora-Vilchis et al. 2012

Oceania Australia 2005-2006 P Passerines 403 1.7 Zamora-Vilchis et al. 2012

Oceania Australia 2004, 2005, H Passerines 188 10.6 Collombelli-Négret and 2006 Kleindorfer 2008

Oceania Australia 2011 P Passerines 299 12.7 Laurance et al. 2013

Oceania Australia 2011 H Passerines 299 27.8 Laurance et al. 2013

Oceania Australia 2005 P Passerines 165 8.5 Beadell et al. 2007

Oceania Australia 2005 H Passerines 165 39.1 Beadell et al. 2007

Oceania Australia 2005 L Passerines 165 1.5 Beadell et al. 2007

Oceania Australia 2008-2011 H Passerines 1005 3.2 Balasubramaniam et al. 2013

Oceania Australia 2011-2013 P, H, L Passerines 97 23.7 This study

South America Brazil 2000-2006, P, H Passerines 1545 35.3 Lacorte et al. 2013 2010

South America Brazil 2007-2009 P, H Passerines 676 42.2 Belo et al. 2011

Oceania French Polynesia 2005 P Passerines 174 1.9 Beadell et al. 2007

South America Guyana 1994-2000 P Passerines 195 42.7 Durrant et al. 2006

South America Guyana 1994-2000 H Passerines 195 23.2 Durrant et al. 2006

Oceania Papua Guinea 2002, 2003 H Passerines 209 31.0 Beadell et al. 2004

Oceania Papua Guinea 2002, 2003 P Passerines 209 10.0 Beadell et al. 2004

Temperate

South America Chile 2003-2005 P, H, L Passerines 760 15.4 Merino et al. 2008

Oceania New Zealand 2010 P Passerines 50 2.0 Baillie and Brunton 2011

Oceania New Zealand 2010 P Passerines 145 16.0 Baillie and Brunton 2011

Oceania New Zealand 2010 P Passerines 116 23.0 Baillie and Brunton 2011

Oceania New Zealand 2010 P Passerines 196 20.0 Baillie and Brunton 2011

Oceania New Zealand 2010 P Passerines 44 3.0 Baillie and Brunton 2011

Oceania New Zealand 2010 P Passerines 41 3.0 Baillie and Brunton 2011

South America Uruguay 2002-2003 P Passerines 322 61.5 Durrant et al. 2006

South America Uruguay 2002-2003 H Passerines 322 14.1 Durrant et al. 2006

South America Venezuela 2004-2005 P, H Passerines 527 41.0 Belo et al. 2012

Table 5.6. Haemosporidian prevalence data of urban areas around the world recovered from selected references. Abbreviations include: Type of Haemosporidian (P: Plasmodium, H: Haemoproteus, L: Leucocytozoon) and P (Prevalence in %).

Continent Country Method Date Haemosporidian P (%) Type of bird N sampled Reference

Oceania Australia Molecular 2011-2013 P/H 4.3 Superb-fairy 46 This study wren

Oceania Australia Molecular 2011-2013 L 17.4 Superb-fairy 46 This study wren

Oceania Australia Molecular 2011-2013 P/H 2.2 Superb-fairy 46 This study wren

Oceania Australia Molecular 2011-2013 L 26.7 Superb-fairy 46 This study wren

South America Brazil Molecular & 2007-2009 P/H 56.2 Passerines 676 Belo et al. 2011 microscopy

South America Brazil Molecular 2007 P/H 6.7 Passerines 119 Lima et al. 2010

Europe Germany Microscopy 2000 L 49.0 Eurasian 106 Geue and Partecke blackbird 2008

Europe Germany Microscopy 2000 H 16.0 Eurasian 106 Geue and Partecke blackbird 2008

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Europe Germany Microscopy 2000 P 6.6 Eurasian 106 Geue and Partecke blackbird 2008

Asia Japan Microscopy 1988-2001 P 1.7 Passerines and 701 Murata 2002 non passerines

Asia Japan Microscopy 1988-2001 L 4.6 Passerines and 701 Murata 2002 non passerines

Oceania New Zealand Molecular 2010 P 3.0 Passerines 46 Baillie and Brunton 2011

Europe Tunisia, Spain, Molecular 2006, 2007 P/H 61.0 Eurasian NA Evans et al. 2009 Netherlands, blackbird UK, Germany, Poland, Czech Republic, Latvia, Estonia

North America USA Microscopy 2003 H 8.0 House finch 757 Hartup et al. 2008

North America USA Microscopy 2003 P 5.0 House finch 757 Hartup et al. 2008

North America USA Microscopy 2001 H 3.0 House finch 282 Hartup et al. 2008

North America USA Microscopy 2001 P 3.0 House finch 282 Hartup et al. 2008

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North America USA Microscopy 2006 H 22.0 Northern 23 Fokidis et al. 2008 Mockingbird

North America USA Microscopy 2006 H 18.0 Curve-billed 23 Fokidis et al. 2008 thrasher

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5.4. Discussion

5.4.1. Are Australian Haemosporidians distinct?

Haemosporidians have been recorded on a wide variety of Australian birds

(MacKerras and MacKerras 1960; Beadell et al. 2007; Averis et al. 2009; Zamora-

Vilchis et al. 2012; Laurance et al. 2013) where the most commonly occurring haematozoa are species of Haemoproteus, Leucocytozoon and Plasmodium. These have a broad geographic distribution and infect a wide range of vertebrate hosts world-wide (Santiago-Alarcón et al. 2012), however information for Australia is still limited (Clark et al. 2014). My results suggest that parasite loads of this parasitic group is low compared to other parts of the world, although more extensive sampling is needed to fully explore this point.

This study provides evidence of the presence of avian Haemosporidians in natural and urban areas of eastern Australia. However, the four new lineages here reported suggest that the diversity of Haemosporidians is currently underestimated and together with other published research, suggests that further sampling ought to be undertaken to elucidate the evolutionary distinctiveness of Australian

Haemosporidian parasites in a global context (Clark et al. 2014). Australian studies carried out in tropical (Beadell et al. 2004; Zamora-Vilchis et al. 2012) and temperate areas of the country (Balasubramaniam et al. 2013) have also revealed several unreported lineages of Haemosporidians as in this study. Four linages were detected in this study but their occurrence is differential. While SFW and RBF had

103 a higher prevalence of Leucocytozoon in natural areas, Haemoproteus was absent in urban areas (Table 5.2).

The absence of enough molecular research of Australian Haemosporidians does not allow us to do further analysis about the diversity of the group. For example, it is unknown if the diversity of lineages found in this study are exclusive to the eastern

Australia because this seems to be the first molecular research of avian

Haemosporidians in the region, so geographic distributions are unknown. However, at the present none of the Leucocytozoon or Haemoproteus lineages here reported have been found occurring in RBF from Tropical Australia mainland or Pacific islands (Beadell et al. 2007). Studies in tropical areas from Oceania have reported around 20 different lineages occurring in this region and perhaps no more than 10 lineages in temperate zones of the continent (Baillie and Brunton 2011;

Balasubramaniam et al. 2013).

In Australia, most studies of avian haemosporidians have been carried out in natural and/or rural environments (Lawrence et al. 1946; MacKerras and

MacKerras 1960; Adlar and O'Donoghue 1998; Adlard et al. 2004; Beadell et al.

2004; Beadell et al. 2007; Collombelli-Négrel and Kleindorfer 2008; Averis et al.

2009; Zamora-Vilchis et al. 2012; Laurance et al. 2013). However new descriptions, new host records and biogeographic extensions of bird haemosporidian parasites still take place (Adlar et al. 2002), suggesting the field is in its infancy. And, as shown in this study, even urban areas actually need better sampling.

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5.4.2. Microscopy versus Molecular techniques for sampling Haemosporidians

While microscopy has been used for Haemoporidian studies and has been considered a powerful tool (Braga et al. 2011), other sensitive techniques are likely to improve the accuracy of prevalence and diversity estimates in natural and urban areas (see Delgado-V. and French 2012). For instance, PCR methods can provide a more sensible means of detecting prevalence and diversity of parasite lineages than microscopy and are particularly important in cases when there are low levels of parasitaemia or when the quality of smears is poor. Low correspondence between microscopy and molecular methods was detected in my study. Whereas the prevalence of SFW and RBF in urban areas was not detected by microscopy, it was

7.1% and 5% respectively using molecular analysis. Additionally, molecular screening in natural areas allowed the detection of Leucocytozoon and

Haemoproteus in SFW and RBF. In Australia, the early research involved microscopy screening to detect haemosporidians (Lawrence 1946), however, the molecular approach has provided a significant expansion in our understanding with new unrecorded lineages in several passerines (Balasubramaniam et al. 2013, this study).

5.4.3. Are prevalence levels similar to other parts of the world?

The literature review found fewer studies in the Southern Hemisphere compared to the

Northern Hemisphere (Balasubramaniam et al. 2013), especially in Australia (Clark et al.

2014), and this review brings this limited work together and complements other reviews

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(Garamszegi 2011; Lacorte et al. 2013) carried out with Northern Hemisphere emphasis.

The review identified that Australia has some of the lowest levels of parasitism, even in cities. There is no clear reason for this scenario since differences between continents

(due to for example the composition and the occurrence of endemic hosts as is experienced in Australia) and inadequate baseline information on parasites from Austral regions may hamper our understanding of patterns of host infestation and parasite diversity.

Continentally, the prevalence of Haemosporidians in natural areas of Oceania seems to follow the same global pattern where prevalence decreases from tropical to temperate regions (Table 5.5). Similarly urban areas with tropical cities have more individuals with Haemosporidians than those sampled in temperate cities

(Table 5.6). It has been suggested that this may be a result of a low abundance of vectors in temperate locations (Balasubramaniam et al. 2013).

5.4.4. Do urban areas have greater prevalence of Haemosporidians?

I detected a lower prevalence of Haemosporidians in urban areas compared to natural sites in Australia, a pattern that is corroborated in other areas around the world (e.g. Evans et al. 2009). What this study suggests is that cities are not areas where birds become seriously infected with parasites. In fact, they may be rather sterile environments. I suspect that changes in prevalence may be due to changes in abundance of vectors to spread these parasites in cities. Blood-sucking dipteran insects are the vectors of the haemosporidians. The primary vectors of avian

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Plasmodium spp. are mosquitoes belonging among others to the genera Culex,

Aedes, Culiseta, Anopheles and Mansonia whereas Haemoproteus parasites are transmitted by blood-sucking biting midges (Culicoides) and louse-flies, and

Leucocytozoon uses blackflies (Simulium spp), or biting midges (Culicoides spp)

(Santiago-Alarcón et al. 2012). I have found a significant lower prevalence of louse-flies in more urbanised areas, a group of flies that is recognised as one of the main vectors of Haemoproteus (Santiago-Alarcón et al. 2012; Delgado-V. and

French unpub data). Therefore, it is imperative to extend sampling efforts for diversity, distribution and abundance of the insect vectors and a concomitant study of infections in both final hosts and vectors in different microhabitats, particularly near water, where the development of vectors might contribute to greater opportunities to be parasitised (Jansen et al. 2009; Balasubramaniam et al. 2013).

In natural areas, small passerines such as Superb-fairy wren, Red-browed finch,

Eastern spinebill and Silvereye were found to be infected with haemosporidians confirming other studies (e.g. Adlar et al. 2004; Collombelli-Négrel and

Kleindorfer 2008; Zamora-Vilchis et al. 2012). Therefore I recognise these birds as potential bird models to study long-term (seasonal) parasitological studies comparing urban and natural areas. Furthermore, other host species sampled and analysed by microscopy in this study need further work to determine if the low prevalence recorded is a result of the poor performance of microscopy or whether these species have low prevalence of parasitic fauna. For example, Kleindorfer et al. (2006) and Laurance et al. (2013) reported Haemoproteus in New holland honeyeaters and Golden whistler, respectively. Absence of blood parasites in these

107 two species in urban areas might be interpretable by the low number of individuals sampled, an actual low prevalence or a result of the use of microscopy to detect presence.

Leucocytozoon in Australia has been found in natural and rural areas (Adlard et al.

2004) in some of the bird species sampled here. Recently, Beadell et al. (2007) found Leucocytozoon parasiting Red-browed finch and Zamora-Vilchis et al.

(2012) reported it infecting Lewin's honeyeater. During this study, RBF and SFW were the only hosts found parasitized by Leucocytozoon in urban areas (Table 5.2), however we need further studies with bigger samples to determine if

Haemosparasites of the genus Leucocytozoon are really absent in urban individuals of most passerines.

5.5. Conclusions

Human-induced changes may threaten wild populations not only through habitat loss, but by disturbing inter and intraspecific interactions. For this reason, it is essential to understand how anthropogenic habitats affect host-parasite dynamics and define the underlying factors that may act to modifying these interactions. With cities growing globally, and urban diversity becoming important in the conservation of biodiversity (Kantsa et al. 2013), a better understanding of the types of parasites, the mechanisms that influence their prevalence and their impact is needed. Understanding the distributions of parasites in an urbanised world can reveal patterns of parasite prevalence within particular habitats and in comparison

108 to other habitats of different habitat structure. Such differences could shed light on landscape- or patch-level biotic or abiotic factors that might influence exposure to these blood parasites.

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CHAPTER SIX: SPATIAL CHANGES IN HEALTH PARAMETERS AND

BODY CONDITION OF URBAN SENSITIVE BIRDS ALONG AN URBAN

GRADIENT

6.1. Introduction

The diversity of wildlife is negatively correlated with human activities and infrastructure derived from urban development (Gaston et al. 2010). However, while some bird species survive and prosper in urbanised areas (Urban Tolerant Birds, UTB hereafter), others are affected by a wide range of anthropogenic activities and infrastructure (Urban Sensitive Birds, USB hereafter) (Parsons et al. 2006). An increase in some kinds of food and water, loss of suitable vegetation, reduced presence of predators, fewer or limited competitive interactions, and a changed microclimate are some of the most cited factors potentially contributing to the inclusion or exclusion of birds in cities (Fokidis et al. 2008), however, they do not explain (or characterise) fully the presence, establishment and success of some native species that thrive in urban environments. A number of other factors that are more cryptic and unstudied may also be important. For example, parasite and pathogen exposure (see previous chapters,

Bradley and Altizer 2006) and susceptibility are factors that may influence urban wildlife (Delgado-V. and French 2012).

Urban habitat fragmentation and associated anthropogenic pollution may make an environment more stressful for wildlife. Human disturbance (Fernández- Juricic 2002), introduced species (Klotz and Kühn 2010), road networks (Delgado-V. 2007, 2014), residential developments (Lussier et al. 2006), and pollution (Burger et al. 2004) are 110 anthropogenic pressures which affect wildlife. Prolonged or frequent increases in the number of stressors in the urban environment could result in lowering of health (such as haematological measures and body condition) which can exacerbate the susceptibility to parasite infections and immune-mediated diseases, resulting in reproductive and behavioural problems (Ruiz et al 2002; Martin and Boruta 2013).

Haematological measures provide useful indicators of physiological status and have long been used as indicators of metabolic activity, disease, stress and nutritional status.

Haematological parameters have been successfully used in combination with other condition measures to document wildlife health differences in altered environments

(Stevenson and Woods 2006). When combined with health indices, haematological condition and morphological measures are strong parameters of relative health status of individuals and populations, and can be used as an indicator of the physiological status of individual birds and how this varies with habitat and behaviour (Brown 1996).

Measuring changes in haematological and body condition are useful in assessing whether birds are under increased stress. Such changes are likely to result in reduced success and persistence of individuals and local populations (Romero 2004). Chronic stress, health and body condition can potentially reveal important patterns in risk of mortality (Romero 2002). Therefore, to understand the decline of birds as a result of urbanisation it is crucial to collect and monitor the health and condition of individuals and populations (Varela et al. 2006), although this has been poorly studied (Delgado-V. and French 2012).

The relationship between habitat fragmentation and health remains largely unexplored

111 even though some recent research has shown that bird health does change with land use

(Maute et al. 2013). No study, to date, has investigated health in passerines occupying

Australian cities. This chapter seeks to describe the body condition along an urban gradient for two USB in Australia: the Red-browed finch and the Superb-fairy wren.

I monitored six haematological and body condition parameters to check if immune system investment and body condition decrease in more urbanised areas for both birds.

Specifically, I tested the hypothesis that since urban areas are considered to offer less optimal conditions, birds in more urbanised areas (such as industrial areas) would have higher leukocyte counts, haematocrit, and H/L ratio. Additionally, I tested if individuals inhabiting less urban developed sites (such as natural national parks) have a higher body mass and haemoglobin concentrations.

6.2. Methods

6.2.1. Field sites

I caught birds at five sites along a gradient of urbanisation within and around

Wollongong, NSW, Australia. Field work was carried out in the Illawarra region, New

South Wales. The Illawarra includes the coastal land mass east of the escarpment between southern Sydney and the Shoalhaven River where four study sites were selected for having different levels of urbanisation (urbanisation index UI based on

Gómez et al. (2008) and Hamer et al. (2012) index (described in chapter 3):

1. Heavily urbanised and industrial areas. These areas are situated in the city and the

112 surrounding industrialised zone. This area was Green House Park (hereafter GHP, 34˚

26’ 31” S 150˚ 53’ 31” E), a small park situated between the industrial and commercial centre of the city which exhibits high traffic streets and scarce trees and bushes, UI =

48.41%.

2. Residential areas and urban parks. These are suburban areas where houses are surrounded by gardens and street trees. Dalton Park (hereafter DP, 34˚ 23’ 47” S 150˚

53’ 55” E) was a city park surrounded by a matrix of house blocks with variable amounts of mowed grass and ornamental shrubs with occasional large trees, UI =

41.83%.

3. Woodland edge. Ecological Research Centre (hereafter ERC, 34˚ 24’ 17” S 150˚ 52’

15” E) was a woodland edge surrounded by human altered habitats (e.g. urban gardens, paddocks and edge habitats), where there is a dense, shrubby understorey, UI = 28.12%;

4. Sub-natural and Natural areas. These include extensive vegetation with low levels of intervention located in National Parks, Nature Reserves, and State Recreation Areas. A

Natural area was the Murramarang National Park (hereafter MNP, 35˚ 32’ 46” S 150˚

21’ 49” E), which is situated ca. 300 km (UI = 1.05%); and Mount Keira (hereafter MK,

34˚ 24’ 48” S 150˚ 51’ 19” E) was a sub-natural area (UI = 12.23%).

I sampled Red-browed finches Neochima temporalis (RBF, hereafter) and Superb-fairy wrens Malurus cyaneus (SFW, hereafter) which were captured along the urban gradient

(see Chapter 4 for details about the biology and natural history of the birds).

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6.2.2. Sampling

Field work was conducted over two years in breeding and non-breeding seasons from

January 2012 to August 2013. Birds were caught using mist-nets and transported in a clean cotton bag to a nearby processing station where they were measured and a blood sample was taken.

6.2.3. Body condition

Individual right tarsus length was divided by individual weight and this result was used as an indicator of condition (Stevenson and Woods 2006). Tarsus length is considered as a general estimate of an individual's size and individuals with a greater amount of body mass for a given size are thought to have greater energy reserves and better body condition (Green 2001).

Bill and head length is the size of the bird’s head and bill taken from the tip of the bill until the base of head. It was measured with a caliper. The bird’s age and sex were recorded if identifiable.

6.2.4. Haematological condition

A blood sample was taken by venipuncture of the brachial vein with a 26 gauge needle.

The quantity of blood taken varied with bird size but it was no more than 2% of the weight of the bird and depending on quantity and quality it was used for analysing haematocrit, haemoglobin, H/L ratio and/or TLC.

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6.2.4.1. Haematocrit levels and Haemoglobin concentrations

Up to 50 microliters was collected into a heparinised tube. At the end of each day of field sampling, blood contained in 50 microliter capillaries was centrifuged for 5 min at

13000 rev/min. Haematocrit was expressed as the percentage of the tube containing packed red cells and it is a measure of the efficiency of oxygen exchange in an individual. It can identify individuals who may be anaemic (Booth and Elliot 2003) and is therefore a useful measure of a bird's nutritional state.

A portion of blood sample was also used to measure haemoglobin concentration using

Portable HemoCue Hb 201+ (Hemocue, Angelholm, Sweden). High Hb can indicate a large blood oxygen carrying capacity, which could enhance metabolic performance (Lill et al. 2013).

6.2.4.2. Heterophil:Lymphocyte ratio and total leucocyte count

Blood was smeared onto a microscope slide and white blood cells identified and counted. These identify the strength of the immune system (Davis et al. 2008).

Heterophyll:Lymphocyte is used as an index of chronic stress (Fokidis et al. 2008) and has been shown to both correlate with corticosteroid levels and reliably reflect chronic stress in various bird species (Davis et al. 2008; Dehnhard et al. 2011). Heterophylls are the primary phagocytic leukocytes which proliferate in response to infections, inflammation and stress, while leukocyte are involved in a variety of immunological functions such as immune defence (Davis et al. 2008). The ratio was calculated by

115 dividing the number of heterophils seen at 80 fields of view under a microscope by the number of lymphocytes seen at the same number of fields. Heterophils and

Lymphocytes were identified according to the criteria of Clarck et al. (2009). Total

Leucocyte Count (TLC) was calculated by counting the sum of all leukocytes observed in 80 fields of view. It informs about health and strength of the immune system in an individual (Davis et al. 2008).

6.2.5. Statistical analysis

Data were tested for normality (Shapiro-Wilk test) and homogeneity of variance

(Levene's test). I normalised Haematocrit levels using log transformations. Body and blood conditions that were not normally distributed even after transformation were compared for each species separately using non-parametric tests (Kruskall-Wallis).

Post-hoc comparisons between sites were multiple compared using Bonferroni correction. In the few cases where conditions were found to be normally distributed

(such as RBF Haematocrit levels in breeding season), parametric tests (such as ANOVA or t-student tests) were used for analysing differences among sites. Wilcoxon rank sum test (or ANOVA if normally distributed) was used to test the influence of season and sex. If no differences were found, all data were pooled and compared along the urbanisation gradient with no discrimination between season or sex. Differences between sexes could not be distinguished in the Red-browed finch so only season and site were included for this species.

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6.3. Results

6.3.1. Body condition

6.3.1.1. Body mass

In RBF, body mass was lower in GHP (p value adjustment method: bonferroni p<0.05)

(Figure 6.1a). In SFW, however, no difference was detected among sites (Kruskal-Wallis rank sum test, Kruskal-Wallis chi-squared = 4.7991, df = 4, p-value = 0.3085) (Figure

6.1b).

6.3.1.2. Head and bill length

RBF exhibited higher values of head and bill length at the area with less urban influence

(MNP). These values were significant higher than in industrial areas (GHP) (p<0.01) and even higher than in subnatural areas (MK) (p<0.01) (Figure 6.2a). Contrary, SFW had no differences among sites (Kruskal-Wallis chi-squared = 3.7406, df = 4, p-value =

0.4422) (Figure 6.2b).

6.3.2. Haematological condition

6.3.2.1. Haemoglobin concentrations

RBF had the highest levels of Haemoglobin in natural areas (MNP) and the lowest in the most urbanised area (GHP); these sites were significant different to other sites along

117 the urbanisation gradient (p<0.05) (Figure 6.3a). No differences among sites for SFW were detected (Kruskal-Wallis chi-squared = 9.1057, df = 4, p-value = 0.05851) (Figure

6.3b).

6.3.2.2. Haematocrit

There were significant differences between seasons (higher in breeding season) for RBF

(t = 2.9242, df = 160, p-value = 0.0040), however, there were no differences among sites within each season (breeding season: ANOVA F= 0.316, p-value= 0.867 [Figure

6.4a], and non breeding season: Kruskal-Wallis chi-squared = 0.912, df = 4, p-value =

0.9228 [Figure 6.4b]). Differences were not found for SFW (Kruskal-Wallis chi-squared

= 0.912, df = 4, p-value = 0.9228 [Figure 6.4c]).

6.3.2.3. H/L ratio

RBFs captured in the industrial area (GHP) had the highest values of H/L ratio, which were significantly different than the average condition in finches from the natural area

(MNP) (p<0.005) (Figure 6.5a). For SFW, there were no differences between sex in non-breeding season (W = 1042.5, p-value = 0.602), however, some significant differences were detected for breeding season. For example, females (Figure 6.5b) and males (Figure 6.5c) presented the highest H/L ratio values in GHP which is significant different from other places such as MK, ERC, and DP (p<0.05).

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6.3.2.4. Total Leukocyte Count

In RBF, one of the lowest values was found at the natural area (MNP) which is significant different with the ones recorded in other study sites (Figure 6.6a). No differences among sites for SFW were detected (Kruskal-Wallis chi-squared = 4.6494, df = 4, p-value = 0.3252) (Figure 6.6b).

Figure 6.1. Variation of average body mass of a. Red-browed finch and b. Superb- fairy wren along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

119

A a ab b ab b 20 22 24 26 28 Bill and head length (mm) MNP MK ERC DP GHP

B a a aaa 25 27 29 31 Bill and head length (mm) MNP MK ERC DP GHP

Figure 6.2. Variation of average head and bill length of a. Red-browed finch and b. Superb-fairy wren along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

120

 1  dL  g   A a b b ab ab 160 180 200 220

MNP MK ERC DP GHP Haemoglobin concentration Haemoglobin concentration  1  dL  g   B a aaaa 120 160 200 240

MNP MK ERC DP GHP Haemoglobin concentration Haemoglobin concentration

Figure 6.3. Variation of average Haemoglobin concentration (g dL-1) of a. Red-browed finch and b. Superb-fairy wren along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

121

A aa a aa 3.75 3.90 4.05 Log (Haematocrit (%)) Log (Haematocrit MNP MK ERC DP GHP

B a a aa a 3.7 3.8 3.9 4.0 4.1

Log (Haematocrit (%)) Log (Haematocrit MNP MK ERC DP GHP

C a

a a a a Haematocrit (%) 35 45 55 65

MNP MK ERC DP GHP

Figure 6.4. Average Haematocrit level of Red-browed finch in a. Breeding and b. Non Breeding season, and c. Superb-fairy wren along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

122

A b ab a ab ab H/L ratio 0.0 1.0 2.0 3.0

MNP MK ERC DP GHP

B b abcd cb acd d H/L ratio 0.0 0.5 1.0 1.5

MNP MK ERC DP GHP

C b ab a

a a H/L ratio 0.0 0.5 1.0 1.5 2.0

MNP MK ERC DP GHP

Figure 6.5. H/L ratio of a. Red-browed finch and b. Superb-fairy wren females and c. males in Breeding season along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

123

A c a b bc b TLC 0123456

MNP MK ERC DP GHP

B a aa a a TLC 012345

MNP MK ERC DP GHP

Figure 6.6. Variation of Total Leukocyte Count of a. Red-browed finch and b. Superb- fairy wren along sites with different degrees of urbanisation development. Different letters indicate significant differences (p<0.05).

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6.4. Discussion

In this study, I examined the relationships of urban development with health status and body condition to explore how these mechanisms might be involved in the decline of some small passerines in urban areas from Australia. I had predicted that if urban areas are contributing to the decline of small passerine birds, evidence of a negative relationship between urban development and health and body condition should be found. Although I sampled several different measures that are commonly used, I only detected significant differences for Red-browed finch. Excepting Haematocrit, I found significant differences in body and blood conditions for 5 out of 6 considered measurements for RBF. In this species, I particularly found a significant decrease in

Haemoglobin concentration as well as a higher H:L ratio in more urbanised areas compared to natural areas, and a general increase of Total Leukocyte Count in more urbanised locations. Contrary, significant differences for SFW were found in just one condition (i.e. H/L ratio) and restricted to breeding season.

Changes in leucocyte profiles and haemoglobin levels can indicate stress that has detrimental effects in some animals living in cities. For example, body and haematological conditions such as Haemoglobin concentrations, can change due to effects of environmental variation, including habitat fragmentation and habitat quality on individual condition in wild passerines (Davis et al. 1998; Hõrak et al. 1998) and small mammals (Johnstone et al. 2011), which can be connected to individual persistence (Dubiec and Cichon 2001) and breeding success (Gustafsson et al. 1994).

Therefore, while some animals seem to adapt to live in disturbed landscapes, others often decline and are less likely to persist in urban landscapes.

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Previous research on Australian mammals has shown that chronic stress due to anthropogenic activity could contribute to a decline of certain mammals since they can cause physiological stress and make them more susceptible to pathogens, diseases and parasites (Johnstone et al. 2010). These authors also found that anthropogenic habitat fragmentation has detrimental effects on several indicators of population health in the agile antechinus (Antechinus agilis) in urban environments. Brearley et al. (2012) found that individual squirrel gliders (Petaurus norfolcensis) had lower hair cortisol levels in forest interiors compared to those living in habitats adjacent to urban edges. These results are consistent with previous studies on other mammals in relation to other non- urban activities and influence such as those carried out in howler monkeys (Aloutta pigra) (Martínez-Mota et al. 2007). In birds, however, studies of the relationship between urbanisation and individual condition have suggested that health parameter might be more variable and complex.

Some studies have reported patterns where urban individuals have lower condition than rural ones (e.g. House sparrow, Liker et al. 2008). Similarly, urban European blackbirds had lower fat reserves than rural birds (Partecke et al. 2005), and rural American crows were substantially larger and presumed healthier than urban crows (Heiss et al. 2009).

Additionally, Ots et. al. (1998) found lower haematocrit levels for birds breeding in urban versus rural habitats. In addition, in urban areas, Rufous-collared sparrows have lower body weight, higher blood glucose concentration, higher proportion of heterophils, lower proportion of lymphocytes, and consequently, a larger H:L stress index, than rural ones (Ruiz et al. 2002). In comparisons with natural habitats, some variable responses have also been shown. Ovenbirds (Seiurus aurocapillus) nesting in

126 continuous forest habitat had higher mass and haematocrit and normal immune system activity that individual in fragmented forests which were lighter, had lower haematocrits, and may have had depressed immune responses (Mazerolle and Hobson

2002). Contrary, others have found that urban individuals are in better condition than their natural or rural conspecifics (Cypher and Frost 1999) or have documented no significant differences in birds between intact and altered habitats (Gavett and Wakeley

1986) suggesting that these effects might vary from species to species according to the energetic and/or social needs of organisms.

One of the factors that may be causing contrasting results is the kind of species investigated. For instance, species which are common in cities with relatively stable or increasing population sizes may be advantaged by increased immunity whereas species which are now rare in urban environments and declining in abundance may be more susceptible. Thus different species of birds may have different susceptibility to parasite load thus influencing their ability to occupy urban centres (Bonier 2012). This research suggests that responses might be variable, even within the group of urban sensitive birds. For example, Chávez-Zichinelli et al. (2013) studied the Canyon Towhee

(Melozone fusca) and Inca Dove (Columbina inca) and found that physiological variables differed between the species and habitat condition. Similarly, authors found that immunoglobulin concentration in C. inca was higher at urban sites, whereas it was lower in M. fusca, therefore they might exhibit differential health effects due to urbanisation processes.

RBF and SFW are considered not to be abundant in urban areas and affected by urbanisation processes. Parsons et al. (2006) found that RBF is about half as common as

127

SFW in urban areas. While SFW occurs in about 25% of gardens, RBF were only recorded in about 11-12%. Data obtained from my research suggests that this may be associated with RBFs being more stressed and having lower body condition.

Health differences recorded in this study might depend on species and life history stage, particularly on their type of diet and foraging strategy. A lower Hb concentration and a higher H/L ratio and TLC values in more urbanised areas for RBF may result from the higher energy demands imposed by the urban habitat. For instance, as RBF mostly feeds on grass seeds, the fragmentation of the habitat in urban zones, as well as the increased area of pavement, infrastructure, and residences in the urban environment will lower food resources and should impose a greater metabolic pressure in these birds. Therefore in urban areas, food supplies are possibly more commonly low for RBF than for SFW, so RBF are restricted to parts of the city where there is adequate supplies of grass seeds.

The idea that urban birds are stressed by food shortage has been hypothesised for other granivorous birds. For example, Chilean rufous-collared sparrows (Zonotrichia capensis) from urban localities had body weights significantly lower than that of rural ones (Ruiz et al. 2002). However, factors other than differences in food availability may account for the changes. An additional factor that may help to explain differences in body mass and health parameters between SFW and RBF is the effect of the presence of some parasites. While I found that Coccidian parasites are more prevalent in urban RBF while urbanisation increases, prevalence is not significantly different along urban gradients for SFW (see Chapter 4).

128

6.5. Conclusions

According to the results, the direction and magnitude of these differences indicate that that while SFW might have a greater resistance to parasite infections, pathogens and diseases in urban areas, the immune system of urban RBF might not have the optimal condition to respond successfully to urban pressures in cities. Also

I consider that health parameters make suspect that RBF is more prone to have higher infestation by parasites since it is in poorer health and body conditions in the urban populations than SFW.

129

CHAPTER SEVEN: CONCLUSION, GENERAL DISCUSSION AND FUTURE

DIRECTIONS

Changes in host-parasite interactions have the potential to be one of the most important consequences of urbanisation. However, this research found that bird- parasite interactions in urban areas are one of the relationships less studied globally, particularly in Australia (Chapter 1), a territory that exhibits a unique evolutionary history of isolation. Consequently there may be unique lineages of parasites with different patterns of prevalence compared to the rest of the world.

In order to increase our understanding of diversity, prevalence, and distribution of parasites in Australia and Oceania, and how bird-parasite relationships may be affected by human induced changes along an urbanisation gradient in New South

Wales, this thesis focused on trying to understand a possible pattern of spatial dynamics of three types of parasites (ectoparasites, Coccidians and

Haemosporidians). These parasites have different biologies and ways of transmission. Additionally, this research sought to describe body and blood conditions, as well as susceptibility along the gradient of their passerine hosts.

The results found that some parasites, such as louse flies and ticks (Chapter 3) as well as haemosporidians (Chapter 5), are less prevalent (or even disappear) while others might be either more prevalent (e.g. Coccidian parasites, Chapter 4) or stable in urban areas

(e.g. lice, Chapter 3). However, this study was unable to detect a general pattern of loss of body or blood condition associated with urban development for the species sampled

(Chapter 6), suggesting that urbanisation effects are complex and might be both site-

130 parasite- and host-specific related. Thus, future avian health surveys and disease controls should be implemented as strong variation and pathogen pressures are known to fluctuate.

As corroborated in this thesis (four new lineages of haemosporidians were found indicating the great potential for Australian bird parasites to be fundamentally unique,

Chapter 5), the insufficient knowledge on Australian parasite fauna urges inventories and systematic monitoring schemes. It is also important to conduct detection of vectors and intermediate hosts since this information does not exist for many of the parasites recorded herein.

Moreover, parasites sampled during this research are not the only parasites that infest bird hosts: there are other groups that should be considered in the future which are more likely to respond differently due to variable environmental conditions (e.g.

Trichomonas). Certainly, the inclusion of molecular diagnosis should be incorporated in urban screening, since intensity of parasite infection could be below the limit of detection via regular techniques.

There is a growing number of tests used to measure variation and possible effects of parasites on avian hosts (Boughton et al. 2011). The list includes behavioural, immunological and physiological tests. In order to gain a comprehensive idea about body condition, immune responses and differential parasitism, a diverse selection of tests should be considered (Pedersen and Fenton 2006).

Urban ecology should articulate its knowledge on species with the study, understanding

131 and conservation of biotic relationships. However, while studies of urbanisation on biodiversity structure and composition have been numerous in recent years, few of them emphasise the effects of urbanisation on ecological and evolutionary processes and how they maintain and conserve biodiversity within urban environments. What it is needed in the future is an integrative field of study stemming from ecological and physiological approaches, using distribution of parasites along urbanisation gradients, and exploring the potential role of parasites and immune status in determining the structure of urban- bird communities.

132

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